Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.     

Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992-996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H(+)-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.     

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

In this paper we report that adeno-associated virus (AAV) genomes inhibit stable transformation by several dominant selectable marker genes upon cotransfection into mouse tissue culture cells. Cotransfection of AAV genomes also inhibited the expression of pSV2cat in transient assays. In both cases, the inhibitory effect was independent of AAV DNA replication but required the AAV p5 and p19 genes, which encode proteins required for AAV DNA replication and regulation of AAV gene expression. Finally, addition of a cloned E4 gene in the transfection experiments partially blocked the AAV-mediated inhibitory activities.     

Novel Tat-encoding bicistronic human immunodeficiency virus type 1-based gene transfer vectors for high-level transgene expression

We describe bicistronic single-exon Tat (72-amino-acid Tat [Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV). Tat72-encoding vectors performed better than Tat86-expressing vectors in gene transfer experiments. CTE-containing vectors, produced in a Rev-independent packaging system, had gene transfer efficiencies nearly equivalent to those produced using a combination RNA transport (CTE and Rev-Rev response element)-based packaging system. The Tat72-encoding vectors could be efficiently transduced into a variety of cell types, showed higher levels of transgene expression than vectors with the simian cytomegalovirus immediate-early or the simian virus 40 early promoter, and provide an alternative to HIV-1 vectors with internal promoters.     

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.     

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 tat protein

The human immunodeficiency virus type 1 Tat protein is a potent activator of viral gene expression and replication. Tat can also affect the expression of cellular genes including cytokines, extracellular matrix proteins, enzymes degrading the basement membrane and cell cycle-related proteins, and can regulate cellular functions such as growth, migration and angiogenesis. In addition, under certain circumstances, Tat may have tumorigenic effects. These activities of Tat appear to be mediated by different mechanisms such as the transactivation of cellular gene expression or the interaction of extracellular Tat with the cell membrane through both receptor-mediated and nonreceptor-mediated interactions. Deregulation of cellular gene expression and function by Tat cause abnormalities which may participate in AIDS pathogenesis and in the development of AIDS-associated disorders.     

FKBP52对小鼠前列腺发育的影响

目的通过FKBP52基因敲除小鼠模型探索FKBP52在小鼠前列腺发育过程中的作用。方法分别对胚胎第17．5天、新生的和出生后3周的野生型和FKBP52基因敲除小鼠的前列腺进行切片HE染色,观察不同发育时期里野生型和FKBP52基因敲除小鼠前列腺发育的异同。结果（1）小鼠前列腺发育的起始不依赖于FKBP52基因的参与;（2）随着胚胎的发育,FKBP52在雄鼠前列腺发育中的作用逐渐显现出来,即FKBP52的缺失会导致前列腺叶发育受阻,最终不能形成成熟的前列腺。结论FKBP52在小鼠前列腺的发育过程中具有重要作用,它不参与前列腺的发育起始过程,但其缺失会导致前列腺发育受阻,即不能形成成熟的前列腺。     

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.     

Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer

In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring beta-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.