Keyun Qing

Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2

Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. Although AAV uses the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We demonstrate here that cell surface expression of HSPG alone is insufficient for AAV infection, and that AAV also requires human fibroblast growth factor receptor 1 (FGFR1) as a co-receptor for successful viral entry into the host cell. We document that cells that do not express either HSPG or FGFR1 fail to bind AAV and, consequently, are resistant to infection by AAV. These non-permissive cells are successfully transduced by AAV vectors after stable transfections with cDNAs encoding the murine HSPG and the human FGFR1. Furthermore, AAV infection of permissive cells, known to express both FGFR1 and the epidermal growth factor receptor, is abrogated by treatment of cells with basic fibroblast growth factor, but not with epidermal growth factor. The identification of FGFR1 as a co-receptor for AAV should provide new insights not only into its role in the life cycle of AAV, but also in the optimal use of AAV vectors in human gene therapy.

Adeno-associated virus 2 co-receptors?-first reply

Qing et al. reply—The conclusion by Qiu et al. that HEp-2 and A431 cells do not express FGFR is wrong. The Muggeridge et al. report they quote clearly shows that the FGFR number per cell is approximately 300. There are few details of how Qiu et al. generated the data presented in their table. The remarkably high multiplicity of infection used in these experiments is not standard, and it is difficult to reconcile their 60% transduction rate for HeLa cells when others have reported that AAV vectors do not transduce these cells well because of the rate-limiting viral second-strand DNA synthesis. Transduction efficiencies of 40% for HEp-2 and 10% for A431, respectively, are cited as proof that these cells can be transduced in the absence of FGFR expression. Yet, as stated above, these cells do indeed express FGFR (ref. 7). Thus, it seems that the analysis of FGFR by Qiu et al. using flow cytometry with a monoclonal antibody is inadequate to draw such a conclusion. We have compared the transduction efficiency of a recombinant AAV-lacZ vector (4 × 10 particles/cell) and found transduction efficiencies in HeLa and 293 cells of approximately 4% and 20%, respectively, and <1% in A431 cells which are known to efficiently bind AAV (ref. 13). The lack of trangene expression in A431 cells has previously been reported to be due to very high levels of expression of the epidermal growth factor receptor (EGFR) protein tyrosine kinase known to limit the viral second-strand DNA synthesis. The observed lack of transduction of M07e cells, which we showed do express FGFR (ref. 1), has previously been shown to be due to lack of expression of heparan sulfate proteoglycan (HSPG), a coreceptor of AAV. The absolute requirement for the deliberate expression of both HSPG and FGFR1 in Raji cells, which are known to lack expression of both of these genes, to render these cells permissive for AAV infection, strongly supports our contention that both HSPG and FGFR1 serve as coreceptors for AAV. Of course, other co-receptors may be used in other cells.

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

ABSTRACT Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Altered Endocytic Processing Enhances Transduction Efficiency in Murine Fibroblasts

ABSTRACT 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 Type 2-Mediated Gene Transfer: Role of Cellular FKBP52 Protein in Transgene Expression

ABSTRACT Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593–1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835–9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused ≈40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

Heat-shock Treatment-mediated Increase in Transduction by Recombinant Adeno-associated Virus 2 Vectors Is Independent of the Cellular Heat-shock Protein 90 *

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in ∼6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.

303. Impaired Nuclear Transport and Virus Uncoating Limit Recombinant Adeno-Associated Virus 2 Vector-Mediated Transduction of Primary Murine Hematopoietic Cells|[ast]|

The transduction efficiency of adeno-associated virus 2 (AAV) vectors varies greatly in different cells and tissues. Whereas muscle and brain cells are transduced efficiently, controversies abound regarding hematopoietic cell transduction by AAV vectors. For human hematopoietic cells, we documented this problem to be related to the extent of AAV receptor expression (J. Virol., 71: 8262–8267, 1997). Murine hematopoietic cells express the receptor, yet are transduced poorly. Using a murine fibroblast cell line, we reported that the lack of transduction was due to inefficient intracellular trafficking of AAV from cytosol into the nucleus (J. Virol., 74: 992–996, 2000), and that treatment with hydroxyurea (HU) facilitated nuclear transport of AAV in these cells (J. Virol., 75: 4080–4090, 2001). In the present studies, we extended these observations to primitive murine hematopoietic cells. Murine c-kit+, lin− hematopoietic cells were transduced with recombinant AAV-lacZ vectors. Forty-eight hrs. post-transduction, nuclear and cytoplasmic fractions were isolated and low Mr DNA samples extracted from these fractions were analyzed on Southern blots. Approximately 85% of AAV genomes were present in the cytoplasmic fraction. However, when donor mice were injected intra-peritoneally with HU at 1 mg/g body weight, 24 hrs. prior to isolation of cells, the extent of AAV genomes detected in the cytoplasmic fraction was reduced to ~40%, with a corresponding increase in the nuclear fraction, which increased to ~60%, indicating that in vivo administration of HU facilitated nuclear transport of AAV. However, when DNA samples were electrophoresed on denaturing gels and analyzed on Southern blots, it was apparent that a significant fraction of the AAV genome present in the nuclear fraction from cells obtained from HU-treated mice was single-stranded. We tested the hypothesis whether single-stranded genomes were derived from virions that failed to undergo uncoating in the nucleus. Primitive hematopoietic cells, with and without HU-administration in vivo, were transduced with recombinant AAV-lacZ vectors, and 48-hrs. post-transduction, equivalent fractions were either mock-treated, or digested exhaustively with DNase I, and analyzed on DNA slot-blots using a lacZ probe. Whereas majority of the signal, which was resistant to DNase I, was detected in the cytoplasmic fraction in cells obtained from untreated mice, as expected, a substantial fraction of the signal in the nuclear fraction in cells obtained from HU-treated mice was also resistant to DNase I, suggesting that although HU facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second-strand DNA synthesis and transgene expression. Thus, the identification of a yet another obstacle — virus uncoating — has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

342. Self-Complementary Adeno-Associated Virus 2 (AAV)-T Cell Protein Tyrosine Phosphatase Vector as a Helper-Virus to Improve Transduction Efficiency of Conventional AAV Vectors in vitro and in vivo

Although adeno-associated virus 2 (AAV) vectors target the liver efficiently, 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, and consequently, transgene expression (J. Virol., 75: 8968–8976, 2001). We have documented that dephosphorylation of FKBP52 by cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV transduction of primary murine hematopoietic cells in TC-PTP-transgenic mice (J. Virol., 77: 2741–2746, 2003). Since TC-PTP cDNA size is within the packaging capacity of self-complementary AAV (scAAV) vectors, which obviate the need for viral-second-strand DNA synthesis, we reasoned that scAAV-TC-PTP vectors, if co-infected with a conventional AAV vector, might serve as a helper-virus for conventional single-stranded AAV vectors. Co-infection of HeLa cells, which are transduced poorly by conventional AAV vectors, at a 1:1 ratio with a conventional AAV-EGFP vector and scAAV vectors containing the wild-type (wt) TC-PTP gene (scAAV-wt-TC-PTP) led to a significant increase in AAV-mediated EGFP transgene expression in vitro. This increase was not observed when scAAV vectors containing a catalytically inactive mutant form of TC-PTP (scAAV-mTC-PTP) were used. We also tested the efficacy of scAAV-TC-PTP vectors in a mouse model in vivo. Approximately 1 × 1011 particles of AAV-EGFP vectors alone, or those ad-mixed with either scAAV-wtTC-PTP, or scAAV-mTC-PTP vectors were injected via the tail-vein into C57BL6 mice. Four weeks-post injection, liver tissues were harvested, sectioned and evaluated for EGFP gene expression. Consistent with previously published reports, little transgene expression occurred in hepatocytes following injection of conventional AAV-EGFP vectors, however, co-injection with scAAV-wt-TC-PTP vectors, but not with scAAV-mTC-PT-PTP vectors, led to a significant increase in EGFP expression in murine hepatocytes in vivo. Low Mr DNA samples were isolated from liver tissues, electrophoresed on 1% alkaline-agarose gels, and analyzed on Southern blots using an EGFP probe. Dimer-length AAV genomes, representing viral second-strand DNA synthesis, were detected in cells from mice co-injected with conventional AAV-EGFP+scAAV-wt-TC-PTP vectors, but not in those injected with AAV-EGFP vector alone, or co-injected with scAAV-mTC-PTP vectors. Cohorts of C57BL6 mice were also injected via the tail-vein either with PBS, or with 1 × 1011 particles of scAAV-wtTC-PTP vectors and sacrificed at the end of 4-weeks. All tissues from all PBS-injected controls, and scAAV-wtTC-PTP vector-injected animals showed no evidence of any toxicity, and no pathological lesions were observed in any organ in any of the animals. 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.