Introduction

Adeno-associated virus type 2 (AAV) is a nonpathogenic human parvovirus, which contains a single-stranded DNA as its genome^1,2. Since single-stranded DNA genomes are transcriptionally inactive, others and we have suggested that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression^{3,4,5,6,7,8,9,10}. We have previously shown that HeLa cells in vitro, and murine hepatocytes in vivo, are transduced poorly by recombinant AAV vectors since AAV genomes fail to undergo second-strand DNA synthesis in these cells^9,10,11. In our previous studies, we have identified that a 52-kDa cellular protein, FKBP52, which binds the immunosuppressant drug FK506^12,13, interacts specifically with the D sequence within the inverted terminal repeat (ITR) of the AAV genome⁷. FKBP52 is phosphorylated at tyrosine residues by the cellular epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), and tyrosine-phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis, leading to inefficient transgene expression^5,6,7,8,9,10. We have also documented that FKBP52 is dephosphorylated at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP)^14,15, which leads to efficient viral second-strand DNA synthesis and a significant increase in AAV-mediated transduction efficiency in established human cell lines as well as in primary hematopoietic stem cells from TC-PTP-transgenic mice in vitro⁸.

Recently, we have also reported that the efficiency of AAV-mediated transgene expression in murine hepatocytes following intravenous (iv) administration of recombinant AAV vectors carrying the enhanced green fluorescent protein (AAV-EGFP) in FKBP52-knockout (FKBP52-KO) mice and in transgenic mice that express the wild-type (wt) TC-PTP (wtTC-PTP-TG) gene was significantly higher than that in nontransgenic normal mice or in transgenic mice that express a mutant TC-PTP (mTC-PTP-TG) gene¹⁶. We documented that the increase in AAV transduction efficiency in hepatocytes in wtTC-PTP-TG and FKBP52-KO mice was due to more efficient viral second-strand DNA synthesis¹⁶.

Since the TC-PTP cDNA size is within the packaging capacity of recently described self-complementary AAV (scAAV) vectors¹⁷, which obviate the need for viral second-strand DNA synthesis, we reasoned that scAAV-TC-PTP vectors, if co-infected with a conventional single-stranded AAV vector, might serve as a helper virus for conventional single-stranded AAV vectors, as expression of TC-PTP from a double-stranded scAAV-TC-PTP would lead to tyrosine dephosphorylation of FKBP52, resulting in a more efficient viral second-strand DNA synthesis of conventional single-stranded AAV vectors. In our current studies, we have examined whether co-infection with a scAAV-TC-PTP vector results in increased transduction efficiency of a conventional single-stranded AAV vector in vitro as well as in vivo. Whether in vivo administration of scAAV-TC-PTP vectors leads to any adverse effects in normal mice has also been examined.

Results

A Novel Strategy to Augment AAV Transduction Efficiency in Cells That Do Not Allow Viral Second-Strand DNA Synthesis

We had previously envisaged that since TC-PTP is a key protein tyrosine phosphatase, whose seemingly harmless deliberate expression in vitro and in vivo can significantly enhance AAV transduction efficiency, we could exploit the use of recently described scAAV vectors¹⁷ carrying the wt TC-PTP gene. The strategy to achieve this objective is depicted in FIG. 1. Tyrosine phosphorylated forms of FKBP52 block viral second-strand DNA synthesis, resulting in poor transgene expression in cell types such as HeLa^4,5,6,7,8,18 and murine hepatocytes^{6,11,19,20,21,22}, which are permissive for AAV infection (Fig. 1A). However, if scAAV-TC-PTP vectors are admixed with a conventional single-stranded recombinant AAV vector prior to transduction, expression of TC-PTP from the scAAV vector, which would not require viral second-strand DNA synthesis, would cause tyrosine dephosphorylation of FKBP52. This would lead to a more efficient second-strand DNA synthesis of the conventional AAV vector, resulting in stable high-efficiency transgene expression (Fig. 1B). We tested this possibility experimentally by generating scAAV vectors containing the wt murine TC-PTP gene (scAAV-wt-TCPTP). scAAV-mTC-PTP vectors, in which the catalytic site of TC-PTP was inactivated by replacing the cysteine residue with a serine residue, were also used as appropriate controls. Conventional AAV-EGFP vectors were admixed with either scAAV-wt-TCPTP or scAAV-mTC-PTP vectors at various ratios and used to transduce HeLa cells in vitro, and primary murine hepatocytes in vivo, as described under Materials and Methods.

Figure 1.

(A) Schematic representation of the lack of transgene expression in cells that are nonpermissive for viral second-strand DNA synthesis of a conventional single-stranded AAV vector due to the presence of tyrosine-phosphorylated forms of FKBP52. (B) Strategy to augment the second-strand DNA synthesis of a conventional AAV vector by co-infection with a scAAV-TC-PTP vector. FKBP52 (F), phosphorylated at tyrosine residues (red symbol), which strongly inhibits the second-strand DNA synthesis of a conventional single-stranded AAV vector, is dephosphorylated at tyrosine residues by TC-PTP (blue half-oval), expressed from the scAAV-TC-PTP vector, which allows more efficient second-strand DNA synthesis of the conventional AAV vector and, consequently, leads to more efficient transgene expression.

Full figure and legend (86K)

Co-infection with scAAV-wtTC-PTP Vectors, but Not with scAAV-mTC-PTP Vectors, Increases the Transduction Efficiency of Conventional Single-Stranded AAV-EGFP Vectors in HeLa Cells in Vitro

Human HeLa cells contain FKBP52 phosphorylated predominantly at tyrosine residues and, as a consequence, are transduced poorly by conventional AAV vectors^5,6,7,8,9. To test the hypothesis that scAAV-wtTC-PTP would lead to more efficient second-strand DNA synthesis of the conventional AAV vector, resulting in stable high-efficiency transgene expression in HeLa cells, we co-infected these cells with a conventional AAV-EGFP vector and scAAV vectors containing the wt TC-PTP gene (scAAV-wtTC-PTP) at a 1:1 ratio. From the data shown in Fig. 2A, it is evident that, whereas mock-infected HeLa cells showed no green fluorescence, only 3% of HeLa cells transduced with the conventional AAV-EGFP vector were EGFP positive, consistent with our previously published reports^5,6,7,8,9. When we co-infected cells with the scAAV-wtTC-PTP vector, the transduction efficiency was augmented about fourfold (Fig. 2B). This increase was not observed when the catalytically inactive vector, a mutant in which the catalytic cysteine residue in the active site in TC-PTP had been replaced with a serine residue (scAAV-mTC-PTP), was used under identical conditions.

Figure 2.

AAV-mediated transgene expression in HeLa cells following transduction with AAV-EGFP vector alone or with co-infection of AAV-EGFP and scAAV-TC-PTP vectors. (A) Transgene expression was detected by fluorescence microscopy 48 h postinfection. Original magnification 50. (B) Quantitative analyses of AAV transduction efficiency in HeLa cells following transduction with AAV-EGFP vector alone or with co-infection of AAV-EGFP and either scAAV-wtTC-PTP or scAAV-mTC-PTP vectors. Images from three visual fields were analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean SD). Analysis of variance was used to compare test results with the control and they were determined to be statistically significant.

Full figure and legend (196K)

Co-injection with scAAV-wtTC-PTP Vectors, but Not scAAV-mTC-PTP Vectors, Increases the Transduction Efficiency of Conventional AAV-EGFP Vectors in Murine Hepatocytes in Vivo

We also tested the efficacy of scAAV-TC-PTP vectors in a mouse model in vivo. We injected approximately 1 10¹¹ particles of recombinant AAV-EGFP vectors alone or those admixed with either scAAV-wtTC-PTP or scAAV-mTC-PTP vectors via the tail vein into normal C57BL6 mice. Four weeks postinjection, we harvested, sectioned, and evaluated liver tissues for EGFP gene expression using fluorescence microscopy. As can be seen in Fig. 3A, consistent with previously published reports, little transgene expression occurred in hepatocytes following injection of the conventional AAV-EGFP vector; however, co-injection with scAAV-wtTC-PTP vectors, but not with scAAV-mTC-PTP vectors, led to an approximately sixfold increase in the transduction efficiency of conventional single-stranded AAV-EGFP vectors (Fig. 3B).

Figure 3.

AAV-mediated transduction of hepatocytes from normal mice injected with AAV-EGFP vector alone or co-injected with either scAAV-wtTC-PTP or scAAV-mTC-PTP vector. (A) Transgene expression was detected by fluorescence microscopy 2 weeks postinjection of 1 10¹¹ AAV-EGFP particles/animal or 1 10¹¹ AAV-EGFP particles + 1 10¹¹ scAAV-TC-PTP particles/animal via the tail vein. (B) Quantitative analysis of AAV transduction efficiency in hepatocytes from normal mice injected with AAV-EGFP vector alone or co-injected with scAAV-TC-PTP vectors. Images were analyzed quantitatively as described in the legend to Fig. 2.

Full figure and legend (188K)

Co-Injection with scAAV-wtTC-PTP Vectors, but Not scAAV-mTC-PTP Vectors, Augments Second-Strand DNA Synthesis of Conventional AAV-EGFP Vectors in Primary Murine Hepatocytes in Vivo

We next examined whether the increased transduction efficiency of conventional AAV-EGFP vectors, when co-injected with scAAV-wtTC-PTP vectors, was due to more efficient viral second-strand DNA synthesis of conventional single-stranded AAV vectors since expression of TC-PTP from a double-stranded self-complementary AAV vector would be expected to result in tyrosine dephosphorylation of FKBP52. We isolated low M_r DNA samples at various times postinjection from liver tissues, electrophoresed them on 1% alkaline-agarose gels, and analyzed them on Southern blots using a ³²P-labeled EGFP DNA probe as previously described^18,23. The results are shown in Fig. 4. As is evident, whereas no signal was detected in mice injected with PBS (lane 1), almost all of the input AAV vector genomes were present as single-stranded (ss) DNA in hepatocytes from mice injected with AAV-EGFP vector alone or co-injected with scAAV-mTC-PTP vectors from 1 to 4 weeks (lanes 2, 3, 5, 6, 8, and 9), similar to that observed in normal mice previously¹⁶ and consistent with the observed low transduction efficiency of recombinant AAV vectors in the murine hepatocytes^{11,19,20,21,22}. Interestingly, a significant fraction of the viral genomes was converted to dimer-length DNA duplex forms (d), which migrated slower on the denaturing gel, 1 week after injection, and most of them were converted to DNA duplex forms 2 and 4 weeks postinjection in hepatocytes from mice co-injected with conventional AAV-EGFP + scAAV-wtTC-PTP vectors (lanes 4, 7, and 10). Thus, the extent of viral second-strand DNA synthesis was consistent with the observed transcriptional activity of the vector genomes^{3,4,5,6,7,8,9,10}.

Figure 4.

Southern blot analysis of AAV second-strand DNA synthesis in hepatocytes from normal mice injected with either PBS or AAV-EGFP vector alone or co-injected with scAAV-TC-PTP vectors. Equivalent amounts of low M_r DNA samples from liver tissues from mock-injected and vector-injected mice were analyzed on a denaturing gel using a ³²P-labeled EGFP probe. ss and d denote the unit-length single-stranded and dimer-length single-stranded AAV genomes, respectively.

Full figure and legend (144K)

TC-PTP Overexpression Is Nontoxic in Mice for up to 13 weeks Post-injection of scAAV-wtTC-PTP Vectors in Vivo

Because we were injecting a helper virus expressing a catalytic enzyme, one question that remained unanswered was whether overexpression of TC-PTP was safe. To evaluate this, we injected cohorts of C57BL6 mice via the tail vein either with PBS or with 1 10¹¹ particles of scAAV-wtTC-PTP vectors and sacrificed them at the end of 4 and 13 weeks, as described under Materials and Methods. We subjected equivalent amounts of total RNA isolated from mock-injected or vector-injected mouse livers to reverse-transcribed PCR (RT-PCR) analyses as described under Materials and Methods. A composite of ethidium bromide-stained gels is shown in Fig. 5. As expected, the expression of TC-PTP mRNA was significantly increased in murine hepatocytes for up to 13 weeks postinjection in three mice from each group that were examined, whereas the -actin mRNA levels showed no significant change.

Figure 5.

RT-PCR analysis of TC-PTP mRNA in murine hepatocytes for up to 13 weeks post-injection of either PBS or 1 10¹¹ particles of scAAV-TC-PTP vectors via tail-vein injections in vivo. Three individual mice from each group were examined.

Full figure and legend (45K)

We also examined whether injection of scAAV-wtTC-PTP vectors led to any adverse effect in normal mice. We examined every organ grossly and clinically. Both PBS-and scAAV-wtTC-PTP vector-injected groups of mice were grossly and clinically normal. There were no signs of irritation at the injection site (tail) and no evidence of intercurrent bacterial or viral infections or malformations. We examined the following tissues microscopically: liver, kidneys, heart, spleen, brain (cerebrum, cerebellum, brain stem), spinal cord, testes, small intestine, and sternum (bone, bone marrow). All tissues from all PBS-injected control and scAAV-wtTC-PTP vector-injected animals showed no evidence of any toxicity, and we observed no pathological lesions in any organs in any of the animals. Microscopic examinations were performed by an experience pathologist certified by the American College of Veterinary Pathologists. A set of representative data shown in Fig. 6 corroborates that TC-PTP overexpression was nontoxic in murine hepatocytes for 4 and 13 weeks post-injection of scAAV-wtTC-PTP vectors in vivo.

Figure 6.

Histopathological analyses of murine hepatocytes 4 and 13 weeks post-injection of either PBS or 1 10¹¹ particles of scAAV-TC-PTP vectors in vivo. Five individual mice from each group were examined. Sections were visualized under a light microscope. Original magnification 320.

Full figure and legend (464K)

Discussion

It has become increasingly clear that AAV vectors transduce different cell and tissue types differentially. Parts of this puzzle have been resolved by gaining a better understanding of the fundamental steps in the life cycle of AAV, which include cell surface receptor-and coreceptor-mediated viral binding and entry^24,25,26, intracellular trafficking^{18,27,28,29,30,31,32}, nuclear transport and uncoating^{23,27,28,31,32,33,34}, and viral second-strand DNA synthesis^{3,4,5,6,7,8,9,10}. We have focused our efforts on augmenting AAV second-strand DNA synthesis by pharmacologic and genetic means. For example, we have reported that treatment of cells that are poorly transduced by AAV vectors with specific inhibitors of cellular EGFR-PTK, which leads to inhibition of phosphorylation of FKBP52 at tyrosine residues, results in a significant increase in AAV second-strand DNA synthesis and, consequently, AAV-mediated transgene expression in vitro^5,6,7,9. We have also shown that dephosphorylation of FKBP52 at tyrosine residues by deliberate overexpression of TC-PTP achieves the same objective in cell lines in vitro as well as in transgenic mice in vivo^8,16. However, both of these approaches have little practical value in gene therapy applications with AAV vectors in humans primarily because of the toxicity associated with the inhibitors of EGFR-PTK to primary cells and because of the improbable prospects of generating TC-PTP-transgenic humans.

The simple approach to delivering the TC-PTP gene via a scAAV vector, which serves as a helper virus for a conventional AAV vector both in vitro and in vivo, as documented in our current studies, bodes well for its potential use with any single-stranded AAV therapeutic vector in human gene therapy. The apparent lack of any toxicity of scAAV-TC-PTP vectors, at least in experimental mice, lends credence to the suggestion that the use of this approach might also be safe. This is further supported by the fact that deliberate overexpression of TC-PTP is also not deleterious in our TC-PTP-transgenic mice, which remain fertile and healthy to more than 1.5 years of age^8,16. Furthermore, given that AAV2 remains the sole serotype vector currently in use in human gene therapy, coupled with the fact that it is also the best characterized serotype in terms of vector toxicology, it is conceivable that this strategy could be employed to augment transgene expression in the liver in clinical trials in patients with hemophilia.

Despite a modest increase in the transduction efficiency of conventional AAV vectors with coadministration with scAAV-wtTC-PTP vectors, there are several reasons for optimism that this efficiency could be further augmented. For example, we used scAAV vectors packaged into AAV serotype 2 capsids (scAAV2-TC-PTP), which undoubtedly competed for the same cellular receptor and coreceptor as the conventional AAV serotype 2 vector (AAV2-EGFP), essentially reducing the effective viral titers. This could easily be circumvented by packaging the scAAV-TC-PTP genomes in AAV serotypes other than 2. In this context, it is noteworthy that a recent report suggests that compared with AAV serotype 2 vectors, AAV vectors based on serotype 8 undergo rapid uncoating much more efficiently in murine hepatocytes³⁴. It is tempting to speculate that scAAV-TC-PTP genomes packaged into AAV serotype 8 (scAAV8-TC-PTP) vectors would be more effective at much lower viral multiplicities of infection, thereby further reducing the risk of any cytotoxicity that might be associated with overexpression of TC-PTP. In view of the broad host range of AAV8 vectors, it is also likely that scAAV8-TC-PTP vectors would be efficacious in a wide variety of tissues. In our current studies, the scAAV-TC-PTP gene was under the control of the Rous sarcoma virus (RSV) promoter. It is likely, therefore, that using tissue-specific promoters to drive the expression of the TC-PTP gene, efficient transduction of a given cell type could be achieved. It should be noted, however, that despite a near-total conversion of the viral single-stranded genomes to their duplex counterparts, the extent of increase in the transgene expression is relatively modest. This implies that additional obstacles to efficient transduction by AAV vectors remain, which underscores the need for further studies to circumvent them.

It should also be noted that although the block to AAV second-strand DNA synthesis can be significantly overcome by the use of scAAV-TC-PTP vectors, it is not complete. Because serine/threonine-phosphorylated forms of FKBP52 undoubtedly inhibit the viral second-strand DNA synthesis⁷, it is important now to identify the putative cellular phosphatase that catalyzes the serine/threonine dephosphorylation of FKBP52, which can then be further exploited by the novel strategy we describe here to realize the full potential of recombinant AAV vectors in their optimal use in human gene therapy.

Materials and methods

Cells, viruses, and plasmids

HeLa, the human cervical carcinoma cell line, was obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained as monolayer cultures in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% newborn calf serum (NCS) and 1% (by volume) of 100 stock solution of antibiotics (10,000 U penicillin + 10,000 g streptomycin). Highly purified stocks of a recombinant AAV2 vector containing the EGFP reporter gene driven by the cytomegalovirus (CMV) immediate-early promoter (CMVp-EGFP) were obtained from Virapur, LLC (CA, USA). Recombinant expression plasmids containing the RSV promoter-driven murine TC-PTP cDNA, either the wild-type (wt TC-PTP) or a mutant, in which the catalytic cysteine residue in the active site had been replaced with a serine residue (C-S mutant TC-PTP), were generously provided by Dr. Michel Tremblay (McGill University, Montreal, Quebec, Canada). The self-complementary AAV2 packaging vector, pLY-4, containing one AAV2 ITR in which the D sequence was replaced by a substitute (S) sequence, was constructed by standard cloning methods using plasmids pXS-23 and pXS-36 described previously³⁵. The RSV promoter-driven murine wt TC-PTP or C-S mutant TC-PTP cDNA was inserted in-between two AAV ITRs in plasmid pLY-4. Recombinant scAAV2 vectors containing the RSV promoter-driven murine wt TC-PTP (scAAV-wtTC-PTP) or C-S mutant TC-PTP (scAAV-mTC-PTP) were generated as described previously³⁶. The scAAV-TC-PTP vector genomes were characterized using alkaline-agarose gel electrophoresis and Southern blot hybridization, and physical particle titers of recombinant vector stocks were determined by quantitative DNA slot-blot analysis as previously described^17,37.

Recombinant AAV2 vector transduction study in vitro

Approximately 1 10⁵ HeLa cells were plated in each well in a 12-well plate and incubated at 37°C for 12 h. Cells were washed once with IMDM and then infected at 37°C for 1 h with mock and 5 10³ particles per cell of recombinant AAV2-wt TC-PTP or C-S mutant TC-PTP vector followed by infection with 5 10³ particles per cell of a recombinant AAV2-EGFP as described previously⁷. Cells were incubated in complete IMDM containing 10% NCS and 1% antibiotics for 48 h. The transduction efficiency was measured by GFP imaging using a Zeiss Axiovert 25 fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY, USA). Images from three visual fields of mock-infected and vector-infected HeLa cells 48 h postinjection were analyzed quantitatively by ImageJ analysis software (NIH, Bethesda, MD, USA). Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean SD). Analysis of variance (ANOVA) was used to compare between test results and the control and they were determined to be statistically significant.

Recombinant AAV2 vector transduction study in vivo

Recombinant AAV2-EGFP vectors or recombinant AAV2-EGFP vectors and recombinant self-complementary AAV2-wt TC-PTP or C-S mutant TC-PTP vectors (at 1:1 ratio) were injected intravenously via the tail vein into C57BL6 mice at 1 10¹¹ virus physical particles per animal. Liver sections from three hepatic lobes of the mock-injected and injected mice 2 weeks after injection were mounted on slides. The transduction efficiency was measured by GFP imaging using a Zeiss Axiovert 25 fluorescence microscope (Carl Zeiss). Images from three visual fields of mock-injected and vector-injected mice 2 weeks postinjection were analyzed quantitatively using ImageJ analysis software (NIH). Transgene expression was assessed as total area of green fluorescence (pixel²) per visual field (mean SD). ANOVA was used to compare between test results and the control, and they were determined to be statistically significant.

Southern blot analysis of AAV2 second-strand DNA synthesis

Equivalent amounts of liver tissues (approximately 0.3 g) from either mock-injected or vector-injected mice were immediately frozen in liquid nitrogen and homogenized by an alloy tool steel mortar and pestle set (Fisher, Chicago, IL, USA). Low M_r DNA samples were isolated and electrophoresed on 1% alkaline-agarose gels followed by Southern blot hybridization using a ³²P-labeled EGFP DNA probe as described previously^18,23.

RT-PCR

Total RNA was isolated from equivalent amounts of liver chunks (approximately 0.1 g) from mock-injected or vector-injected mice using a Trizol RNA isolation kit (Invitrogen Life Technologies, Carlsbad, CA, USA) and was treated with DNase I. Equivalent amounts of total RNA were reverse transcribed followed by PCR analysis with the following primer pairs: 5'-CGGTTAAATGTGCACAGTACTGGCC-3' and 5'-CTACAACGAGAAGGTGCGAGAGC-3' for TC-PTP, 5'-CCTATGGCGCAGCAGGCA-3' and 5'-TCTGCGGATGATCCCGCC-3' for FKBP52, and 5'-ATGAAGATCCTGACCGAGCG-3' and 5'-TACTTGCGCTCAGGAGGAGC-3' for -actin. Equivalent amounts of amplified DNA products were electrophoresed on 2% agarose gels.

Toxicology studies

Two sets of five 4-week-old normal C57BL6 male mice were injected via the tail vein either with PBS (control) or with 1 10¹¹ scAAV-wtTC-PTP vectors (treated). The rest of the studies were outsourced to Micagenix, Inc. (Greenfield, IN, USA). One set of control and treated mice was sacrificed 4 weeks postinjections, and the second set was sacrificed 13 weeks postinjections. Each mouse was given an external and an internal gross examination. Tissues and organs were collected from each set and placed in 10% buffered formalin for fixation and processed for microscopic examination. The following tissues were examined microscopically: liver, kidneys, heart, spleen, brain (cerebrum, cerebellum, brain stem), spinal cord, testes, small intestine, sternum (bone, bone marrow), and cross section of tail (iv site of injection). Microscopic examinations were performed by a pathologist certified by the American College of Veterinary Pathologists, who has 28 years of experience in toxicological pathology.

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Acknowledgements

We thank Dr. Michel Tremblay for generously providing the TC-PTP expression plasmids and Dr. Jacqueline A. Hobbs for a critical review of the manuscript. This research was supported in part by Public Health Service Grants R01 HL-63169 (to M.C.Y.) and R01 EB-002073, HL-65570, and HL-76901 (to A.S.) from the National Institutes of Health (NIH). K.A.W.-K. was supported by NIH Training Grant T32 HL-07910 (to A.S.).