Browsing by Author "Qing, Keyun"

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    Adeno-associated Virus 2-Mediated Transduction and Erythroid Lineage-Restricted Long-Term Expression of the Human β-Globin Gene in Hematopoietic Cells from Homozygous β-Thalassemic Mice
    (Elsevier, 2001-06) Tan, Mengqun; Qing, Keyun; Zhou, Shangzhen; Yoder, Mervin C.; Srivastava, Arun; Microbiology and Immunology, School of Medicine
    Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human β-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1+, lin− hematopoietic stem cells from homozygous β-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human β-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW41/41 anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human β-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human β-globin gene sequences were present at ∼1 copy per cell. The efficiency of the human β-globin gene expression was determined to be up to 35% compared with the murine endogenous β-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human β-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human β-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of β-thalassemia and sickle-cell disease.
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    Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response
    (Mary Ann Liebert, Inc., 2020-05) Kwon, Hyung-Joo; Qing, Keyun; Ponnazhagan, Selvarangan; Wang, Xu-Shan; Markusic, David M.; Gupte, Siddhant; Boye, Shannon E.; Srivastava, Arun; Pediatrics, School of Medicine
    A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.
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    Infection of Purified Nuclei by Adeno-associated Virus 2
    (Elsevier, 2001-10) Hansen, Jonathan; Qing, Keyun; Srivastava, Arun; Microbiology and Immunology, School of Medicine
    Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, requires co-infection with a helper virus for its optimal replication. Although AAV possesses a broad host range, certain cell types lack the machinery necessary for efficient entry into the cell and intracellular trafficking of AAV into the nucleus, where the viral second-strand DNA synthesis must occur before gene expression. We have demonstrated that in less-permissive mouse fibroblasts, the virus fails to transport to the nucleus due to altered endocytic processing. However, relatively little is known about the intracellular site of viral uncoating and transport of the virion across the nuclear envelope. Here, we provide evidence that AAV can efficiently enter intact nuclei purified from both permissive and less-permissive cell types. Furthermore, entry into the nucleus is time- and temperature-dependent, but is not saturable and seems to occur independently of the nuclear pore complex. We also demonstrate that purified nuclei contain all of the machinery necessary for uncoating and viral second-strand DNA synthesis even in the absence of a helper virus. These studies provide new insights into the basic biology of AAV and may also have implications for the optimal use of AAV vectors in human gene therapy.
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    Self-complementary Adeno-associated Virus 2 (AAV)–T Cell Protein Tyrosine Phosphatase Vectors as Helper Viruses to Improve Transduction Efficiency of Conventional Single-Stranded AAV Vectors in Vitro and in Vivo
    (Elsevier, 2004-11-01) Zhong, Li; Chen, Linyuan; Li, Yanjun; Qing, Keyun; Weigel-Kelley, Kirsten A.; Chan, Rebecca J.; Yoder, Mervin C.; Medicine, School of Medicine
    Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to ∼5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.