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Metal elements are essential components of approximately half of all cellular proteins, and approximately one-third of all known enzymes thus far are metalloenzymes. Several cellular proteins and enzymes undoubtedly impact the transduction efficiency of recombinant adeno-associated virus (AAV) vectors, but the precise role of metal ions in this process has not been studied in detail. In the present studies, we systematically evaluated the effects of all 10 essential metal ions (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc) on the transduction efficiency of AAV vectors. We report herein that five essential metal ions (iron, magnesium, manganese, molybdenum, and sodium) had little to no effect, and calcium strongly inhibited the transduction efficiency of AAV2 vectors. Whereas copper and potassium increased the transduction efficiency by ∼5-fold and ∼2-fold, respectively, at low concentrations, both essential metals were strongly inhibitory at higher concentrations. Calcium also inhibited the transduction efficiency by ∼3-fold. Two metal ions (cobalt and zinc) increased the transduction efficiency up to ∼10-fold in a dose-dependent manner. The combined use of cobalt and zinc resulted in more than an additive effect on AAV2 vector transduction efficiency (∼30-fold). The transduction efficiency of AAV serotypes 1 through 6 (AAV1–AAV6) vectors was also augmented by zinc. Similarly, the transduction of both single-stranded (ss) and self-complementary (sc) AAV3 vectors was enhanced by zinc. Zinc treatment also led to a dose-dependent increase in expression of a therapeutic protein, the human clotting factor IX (hF.IX), mediated by scAAV3 vectors in a human hepatic cell line. This simple strategy of essential metal ion-mediated enhancement may be useful to lower the dose of AAV vectors for their optimal use in human gene therapy., Graphical Abstract, AAV vectors undoubtedly interact with host cell proteins during transduction. Since nearly half of all cellular proteins, and approximately one-third of all known enzymes, are known to be metalloproteins, Rambhai et al. systematically evaluated the effects of all 10 essential metal ions and identified zinc and cobalt as significantly enhancing the transduction efficiency of AAV vectors.

We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as β-thalassemia and sickle cell disease.

Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT’s short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.

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.

Cathepsin S (Cat S) plays an important role in many pathological conditions, including abdominal aortic aneurysm (AAA). Inhibition of Cat S may provide a new treatment for AAA. To date, several classes of Cat S inhibitors have been reported, many of which form covalent interactions with the active site Cys25. Herein, we report the discovery of a novel series of noncovalent inhibitors of Cat S through a medium-throughput focused cassette screen and the optimization of the resulting hits. Structure-based optimization efforts led to Cat S inhibitors such as 5 and 9 with greatly improved potency and drug disposition properties. This series of compounds binds to the S2 and S3 subsites without interacting with the active site Cys25. On the basis of in vitro potency, selectivity, and efficacy in a CaCl2-induced AAA in vivo model, 5 (LY3000328) was selected for clinical development.

Recombinant adeno-associated virus 2 (AAV) vectors encapsidate single-stranded genomes of either polarity equally frequently in separate mature virions. Because viral genomes of either polarity are transcriptionally inactive, both the failure to undergo viral second-strand DNA synthesis and the failure to undergo DNA strand annealing have been proposed as possible reasons to account for the observed low efficiency of transgene expression. We compared the transduction efficiencies of conventional AAV vectors containing both [-] and [+] polarity genomes with those containing either the [-] or the [+] polarity genomes, in vitro as well as in vivo. We document that the transduction efficiency of single-polarity AAV vectors is significantly enhanced by (i) co-infection with adenovirus; (ii) small interfering RNA (siRNA)-mediated down-modulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which inhibit AAV second-strand DNA synthesis; (iii) over-expression of a cellular protein tyrosine phosphatase, T cell protein tyrosine phosphatase (TC-PTP), which catalyzes tyrosine-dephosphorylation of FKBP52; and (iv) deliberate over-expression of TC-PTP, or the absence of FKBP52, respectively, in TC-PTP-transgenic mice and in FKBP52-knockout mice. These data confirm that viral second-strand DNA synthesis, rather than DNA strand annealing, is the rate-limiting step in efficient transduction by AAV vectors. This finding has implications in the use of these vectors in human gene therapy.

Cocaine- and amphetamine-regulated transcript (CART) peptide is a neuro-peptide implicated in the regulation of energy homeostasis. CART mRNA and its encoded peptide have been found in many brain regions that regulate energy balance. To investigate the effects of chronic central expression of CART in the diet-induced-obese (DIO) rats, we constructed and packaged recombinant adeno-associated virus (AAV) 2 vector containing rat CART cDNA and a reporter gene encoding green fluorescence protein (AAV-rCART-hrGFP) driven by the CMV promoter. Approximately 1x10(11) particles of AAV-rCART-hrGFP or control vector AAV-IRES-hrGFP (AAV-hrGFP in short) were injected through intracerebroventricular (ICV) cannulas into adult male DIO Long-Evans rats. Throughout the 7-month study period, AAV-rCART-hrGFP-injected rats had a significantly decreased food intake and body weight gain when compared with those of AAV-hrGFP-injected rats. AAV-rCART-hrGFP injection also modulated hyperphagia following a 24-hour fasting in these rats. Body composition analysis indicated that decreased body weight gain was due to reduction in lean body mass while fat mass was not affected. Expression of green fluorescence protein (GFP) suggested that recombinant AAV virions are located at cells surrounding the third ventricle and medial eminence. Our results demonstrate that chronic expression of CART in brain can modulate energy intake in diet-induced-obese rats. The CART pathway plays an important role in body mass regulation in DIO rats.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Objective: To investigate stearoyl-coenzyme A desaturase (SCD) 1 expression in obesity-prone C57BL/6 mice and in obesity-resistant FVB mice to explore the relationship of SCD1 expression and susceptibility to diet-induced obesity. Research Methods and Procedures: Nine-week-old C57BL/6 and FVB mice were fed either a high- or low-fat diet for 8 weeks. Body weight and body composition were measured before and at weeks 4 and 8 of the study. Energy expenditure was measured at weeks 1 and 5 of the study. Hepatic SCD1 mRNA was measured at 72 hours and at the end of study. Plasma leptin and insulin concentrations were measured at the end of study. Results: When C57BL/6 mice were switched to a calorie-dense high-fat diet, animals gained significantly more body weight than those maintained on a low-calorie density diet primarily due to increased fat mass accretion. Fat mass continued to accrue throughout 8 weeks of study. Increased calorie intake did not account for all weight gain. On the high-fat diet, C57BL/6 mice decreased their energy expenditure when compared with mice fed a low-fat diet. In response to 8 weeks of a high-fat diet, SCD1 gene expression in liver increased >2-fold. In contrast, feeding a high-fat diet did not change body weight, energy expenditure, or SCD1 expression in FVB mice. Discussion: Our study showed that a high-fat hypercaloric diet increased body adiposity first by producing hyperphagia and then by decreasing energy expenditure of mice susceptible to diet-induced obesity. Consumption of a high-fat diet in species predisposed to obesity selectively increased SCD1 gene expression in liver.

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.

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.

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.

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

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.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea 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. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 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.

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.

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 approximately 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 in22-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.

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 beta-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human beta-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW(41/41) anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human beta-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human beta-globin gene sequences were present at approximately 1 copy per cell. The efficiency of the human beta-globin gene expression was determined to be up to 35% compared with the murine endogenous beta-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 beta-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 beta-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 beta-thalassemia and sickle-cell disease.

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 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector 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 recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593–1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.

The p Sub 201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34 + primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1 + lin − primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

Although the adeno-associated virus type 2 (AAV) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that AAV infection of human cells is receptor mediated (S. Ponnazhagan et al., J. Gen. Virol. 77:1111-1122, 1996). In the present studies, we investigated the ability of AAV to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. Bone marrow-derived CD34+ cells from 12 hematologically normal volunteer donors were infected with a recombinant AAV containing the beta-galactosidase gene under the control of the cytomegalovirus immediate-early promoter (vCMVp-lacZ). Whereas 15 to 80% of the cells from approximately 50% of the donors showed various levels of lacZ gene expression, the expression was undetectable in cells from the remaining donors. However, if cells from both sets of donors were stimulated with various combinations of cytokines to induce differentiation into myeloid and lymphoid lineages following AAV infection, then the level of expression of the transduced gene increased up to 20-fold over a period of 14 days. The results of virus-binding assays suggested that the observed difference between the two groups was due to the differential susceptibility of CD34+ cells to AAV infection rather than to differences in transcription and translation of the transduced gene. To corroborate these results, CD34+ cells from the two donor groups, KB (human nasopharyngeal carcinoma) cells, and M07e (human megakaryocytic leukemia) cells were infected with vCMVp-lacZ. KB cells served as a positive control for AAV infection, and M07e cells served as a negative control. Whereas abundant hybridization to the single-stranded viral DNA on Southern blots was detected in KB and CD34+ cells that were positive for lacZ gene expression, little activity was detected in M07e and CD34+ cells that did not show expression of the lacZ gene. These results suggest that the levels of expression of the putative cellular receptor for AAV vary widely in CD34+ cells from different donors. These studies have implications for the potential use of AAV vectors in human gene therapy involving primary human primitive hematopoietic stem and progenitor cells.

Cocaine- and amphetamine-regulated transcript (CART) peptide is a neuro-peptide implied in the regulation of energy homeostasis. CART mRNA and its encoded peptide have been localized in many brain regions that regulate energy balance. To study the physiological function of CART in the diet-induced obese (DIO) rats, we constructed and packaged recombinant adeno-associated virus 2 (AAV) vector containing CMV promoter driven rat CART cDNA and a reporter gene hrGFP (AAV-rCART-hrGFP). Approximately 1|[times]|1011 particles of AAV-rCART-hrGFP or control vector AAV-IRES- hrGFP were injected through intra-cerebro-ventricular (ICV) into 100 days old male DIO Long-Evans rats (n=9). Starting on three days post-injection, AAV-rCART-hrGFP-injected rats significantly decreased food intake and body weight gain when compared with that of AAV-IRES-hrGFP-injected rats. AAV-rCART-hrGFP injection modulated hyperphagia following overnight fasting in these rats. Body composition analysis indicated that decreased body weight was due to reduction in lean body mass while fat mass was not different from the control animals. Expression of green fluorescence protein (GFP) suggested that recombinant AAV virions are located at cells around the 3rd ventricle, paraventricle nuclei and medial eminence, external. Our results demonstrate that chronic over-expression of CART in brain can decrease body lean mass by inducing negative energy balance in rats.

Top of pageAbstract Hematopoietic stem cells (HSCs) are attractive targets for gene therapy of a number of diseases. Recent studies have demonstrated the therapeutic potential of recombinant retroviral vectors in hematopoietic stem cell gene transfer in children with severe combined immunodeficiency by, but subsequent development of leukemia in three treated children hasunderscored the need to develop alternative vectors. Recombinant AAV vectors have to date not been associated with any malignant disease and have gained attention as potentially useful vectors. Although conflicting data exist with regard to hematopoietic stem cell transduction by AAV serotype 2 (AAV2) vectors, additional serotypes vectors, AAV1-5, have not been evaluated for their efficacy for hematopoietic stem and progenitor cell transduction. In the present studies, we evaluated the efficacy of AAV serotype vectors 1 through 5 containing the CMV promoter-driven EGFP gene in hematopoietic progenitor cell assays in vitro, as well as following bone marrow transplantation in lethally-irradiated recipient syngeneic mice in vivo. Sca1+, c-kit+, lin- hematopoietic cells following either-mock-infection, or infection with serotype vectors were plated in methylcellulose cultures and hematopoietic colonies expressing the transgene were evaluated 7 days-post-infection. Approximately 5% of the colonies in cultures infected with AAV1 were EGFP-positive. All other serotype vectors elicited no transgene expression. Co-infection of AAV1 with self-complementary AAV vectors containing the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increase the transduction efficiency to |[sim]|20%. Sca1+, c-kit+, lin- cells following either-mock-infection, or infection with serotype vectors|[plusmn]|scAAV-TC-PTP vectors were also injected via tail-vein into lethally-irradiated syngeneic recipient mice. Six months post-transplantation, primary recipient mice were sacrificed and transgene expression was assessed in peripheral blood cells. Approximately 8% of cells from mice transplanted with cells co-infected with AAV1+scAAV-TC-PTP vectors were EGFP-positive. Transduction efficiency of all other serotype vectors was low. Bone marrow mononuclear cells from primary recipient mice transplanted with cells co-infected with AAV1+scAAV-TC-PTP vectors were also used to transplant lethally-irradiated syngeneic mice, and 4 months post-transplantation, |[sim]|3% of bone marrow mononuclear cells were EGFP-positive. These results document that AAV1 is by far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Additional studies involving scAAV genomes and cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene transfer as well as in human gene therapy.

We have reported that transduction efficiency of recombinant AAV vectors is impaired in a murine fibroblast cell line as well as in primary murine hematopoietic stem/progenitor cells (J. Virol., 74: 992, 2000; Hum. Gene Ther., 15: 1207, 2004) because AAV fails to traffic efficiently into the nucleus. We have also reported that a cellular protein, FKBP52, phosphorylated at tyrosine or serine/threonine residues, inhibits viral second-strand DNA synthesis and limits transgene expression (J. Virol., 75: 8968, 2001), and that in hematopoietic stem/progenitor cells from transgenic mice overexpressing TC-PTP, a cellular protein tyrosine phosphatase, AAV transduction efficiency is significantly augmented (J. Virol., 77: 2741, 2003). A significant augmentation is also achieved following treatment of these cells with hydroxyurea (HU) (J. Virol., 75: 4080, 2001; Hum. Gene Ther., 15: 1207, 2004), because HU facilitates nuclear transport of AAV in these cells. In FKBP52-knockout (FKBP52-KO) mice, however, following HU-treatment, transduction efficiency in hematopoietic stem/progenitor cells was significantly less pronounced than that from TC-PTP-TG mice (Hum. Gene Ther., 15: 1207-1218, 2004). To further explore whether FKBP52 plays an additional role(s) in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) from wild-type (WT), FKBP52-heterozygous (HE), and FKBP52-KO (KO) mice. As expected, AAV-mediated transgene expression was low in WT MEFs, and was not significantly increased in HE or KO MEFs. The low transduction efficiency was not due to lack of expression of heparan sulfate proteoglycan and fibroblast growth factor receptor 1, the cellular receptor and co-receptor, respectively, for AAV. Southern blot analyses further confirmed that AAV could efficiently enter MEFs from each of three genotypes. However, whereas HU-treatment increased AAV transduction efficiency in WT MEFs |[sim]|25-fold, the transduction efficiency in KO MEFs was increased only |[sim]|4 fold. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU-treatment increased transduction efficiency |[sim]|23 fold in WT MEFs, but only |[sim]|4 fold in KO MEFs. Thus, the differential transduction efficiency following HU- treatment was independent of viral second-strand DNA synthesis. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU-treatment significantly increased transduction efficiency of scAAV vectors compared with mock-transfected KO cells. Taken together, these studies suggest that in addition to inhibiting viral second-strand DNA synthesis, FKBP52, being a cellular chaperone protein, may facilitate intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

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.

Adeno-associated virus 2 (AAV) vectors are in use in clinical trials for gene therapy of hemophilia B. We have reported that following tail-vein injections, AAV vectors selectively target the liver in mice (Gene, 190: 203–210, 1997). Although 100% of hepatocytes can be targeted by AAV vectors, the transgene expression is limited to ~5% of hepatocytes. Because the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have documented that a cellular protein, designated FKBP52, in its phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, transgene expression in non-hepatic cells (J. Virol., 75: 8968–8976, 2001; J. Virol., 77: 2741–2746, 2003). To evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice over-expressing the cellular T cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. Approximately 1×1011 particles of recombinant AAV-EGFP vectors were injected via the tail-vein in cohorts of normal C57BL6, TC-PTP transgenic (TC-PTP-TG), and FKBP52-knockout (FKBP52-KO) mice. Liver tissues were harvested 1-, 2-, and 4-weeks post-injections and sections were analyzed for transgene expression using confocal microscopy. Consistent with previously published reports, whereas ~5% of hepatocytes from normal mice were transduced, the transduction efficiency increased ~15-fold and ~4-fold, 1-week post-injection; ~17-fold and ~7-fold, 2-weeks post-injection; and ~16-fold and ~12-fold, 4-weeks post-injection in TC-PTP-TG and FKBP52-KO mice, respectively. We also examined whether the increase in AAV transduction efficiency in hepatocytes from TC-PTP-TG and FKBP52-KO mice was due to more efficient strand-annealing or viral second-strand DNA synthesis. Low Mr DNA samples isolated from liver tissues 1-, 2-, and 4-weeks post-injection were electrophoresed on denaturing gels and analyzed on Southern blots. Whereas almost all of the input AAV vector genomes were present as single-stranded DNA in hepatocytes from control mice, a significant fraction of the viral genomes was converted to dimer-length DNA forms 1-week after injection, and most of them were converted to dimer length forms 2- and 4-weeks post-injection in TC-PTP-TG mice as well as in FKBP52-KO mice, albeit at a lower level. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy,1

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.