Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage.

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

Pre-Existing Immunity to a Nucleic Acid Contaminant-Derived Antigen Mediates Transaminitis and Resultant Diminished Transgene Expression in a Mouse Model of Hepatic Recombinant Adeno-Associated Virus-Mediated Gene Transfer.

Liver injury with concomitant loss of therapeutic transgene expression can be a clinical sequela of systemic administration of recombinant adeno-associated virus (rAAV) when used for gene therapy, and a significant barrier to treatment efficacy. Despite this, it has been difficult to replicate this phenotype in preclinical models, thereby limiting the field's ability to systematically investigate underlying biological mechanisms and develop interventions. Prior animal models have focused on capsid and transgene-related immunogenicity, but the impact of concurrently present nontransgene or vector antigens on therapeutic efficacy, such as those derived from contaminating nucleic acids within rAAV preps, has yet to be investigated. In this study, using Ad5-CMV_GFP-immunized immunocompetent BALB/cJ mice, and a coagulation factor VIII expressing rAAV preparation that contains green flourescent protein (GFP) cDNA packaged as P5-associated contaminants, we establish a model to induce transaminitis and observe concomitant therapeutic efficacy reduction after rAAV administration. We observed strong epitope-specific anti-GFP responses in splenic CD8+ T cells when GFP cDNA was delivered as a P5-associated contaminant of rAAV, which coincided and correlated with alanine and aspartate aminotransferase elevations. Furthermore, we report a significant reduction in detectable circulating FVIII protein, as compared with control mice. Lastly, we observed an elevation in the detection of AAV8 capsid-specific T cells when GFP was delivered either as a contaminant or transgene to Ad5-CMV_GFP-immunized mice. We present this model as a potential tool to study the underlying biology of post-AAV hepatotoxicity and demonstrate the potential for T cell responses against proteins produced from AAV encapsidated nontherapeutic nucleic acids, to interfere with efficacious gene transfer.

Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.

Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps.

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.

Bortezomib inhibits angiogenesis and reduces tumor burden in a murine model of neuroblastoma.

BACKGROUND: Bortezomib is a proteasome inhibitor with pleiotropic antitumor activity. Here we investigate the antiangiogenic and antitumor efficacy of bortezomib against neuroblastoma both in vitro and in a murine model of localized and disseminated disease. METHODS: In vitro activity of bortezomib was assessed by evaluating its effect on cell proliferation and cell cycle status. Localized tumor burden was followed with caliper measurements and total-body bioluminescence in mice with disseminated disease. The antiangiogenic activity was evaluated with immunohistochemistry and human vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay on tumor protein extracts. RESULTS: Bortezomib treatment resulted in dose and time-dependent decreases in cell proliferation and resulted in cell cycle arrest. In vivo, bortezomib restricted tumor growth in a model of localized disease and decreased bioluminescence in mice with disseminated disease. That decreased bioluminescence reflected decreased tumor burden was confirmed at necropsy by assessing disease in specific organs. In addition, treatment resulted in a decrease in intratumoral vessel counts and reduced tumor VEGF expression. CONCLUSION: Bortezomib shows significant activity against neuroblastoma in vitro, and it inhibits tumor growth and angiogenesis in vivo. These results suggest that clinical studies of bortezomib are warranted for the treatment of this difficult disease.

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.

With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.

HIF-1α activation mediates resistance to anti-angiogenic therapy in neuroblastoma xenografts.

INTRODUCTION: The anti-tumor activity of angiogenesis inhibitors is often limited by the development of resistance to these drugs. Here we establish HIF-1α as a major factor in the development of this resistance in neuroblastoma xenografts. METHODS: Neuroblastoma xenografts were established by injecting unmodified SKNAS or NB-1691 cells (2 × 10(6) cells), or cells in which HIF-1α expression had been knocked down with shRNA, into the retroperitoneal space of SCID mice. Treatment of established tumors included bevacizumab (5mg/kg q2wk), sunitinib (40 mg/kg qd), or topotecan (0.5mg/kg qd) alone or in combination for a total of two weeks. RESULTS: NB-1691 xenografts showed no difference in relative growth in HIF-1α knockdowns compared to control tumors (73.33 ± 7.90 vs 79.94 ± 6.15, p=0.528). However, HIF-1α knockdowns demonstrated relative final volumes that were significantly lower than unmodified tumors when both were treated with bevacizumab (35.88 ± 4.24 vs 53.57 ± 6.61, p=0.0544) or sunitinib (12.46 ± 2.59 vs 36.36 ± 4.82, p=0.0024). Monotherapy of unmodified xenografts with bevacizumab, sunitinib, or topotecan was largely ineffective. Relative final volumes of NB-1691 xenografts were significantly less in cohorts treated with sunitinib+topotecan (4.78 ± 0.77 vs 39.17 ± 2.44 [sunitinib alone], p=0.011) and bevacizumab+topotecan (13.63 ± 1.55 vs 48.16 ± 9.94 [bevacizumab alone], p=0.014). CONCLUSION: Upregulation of HIF-1α appears to be a significant mechanism of resistance to antiangiogenic therapies in neuroblastoma. Suppressing HIF-1α with low-dose topotecan potentiates the effects of the antiangiogenic drugs in a mouse model.

Rapamycin increases neuroblastoma xenograft and host stromal derived osteoprotegerin inhibiting osteolytic bone disease in a bone metastasis model.

PURPOSE: Osteoprotegerin (OPG) is a decoy receptor for the Receptor of NF-κB (RANK) ligand that can inhibit osteoclastogenesis. Previous studies have suggested that Mammalian Target of Rapamycin (mTOR) inhibition upregulates OPG production. We tested the hypothesis that the mTOR inhibitor rapamycin could inhibit neuroblastoma bone metastases through its action on OPG. EXPERIMENTAL DESIGN: An orthotopic model of bone metastasis was established. Mice with established disease were subsequently treated with rapamycin (5mg/kg IP daily) or vehicle control (DMSO 1:1000). X-rays were obtained twice a week to detect pathologic fractures. Serum OPG levels were measured by ELISA after two weeks of treatment. RESULTS: Mice with bone disease receiving rapamycin had increased serum levels of OPG in the CHLA-20 mice compared to controls (36.89 pg/mL ± 3.90 vs 18.4 pg/mL ± 1.67, p=0.004) and NB1691 tumor-bearing groups (46.03 ± 2.67 pg/mL vs 17.96 ± 1.84pg/mL, p=0.001), and a significantly longer median time to pathologic fractures with CHLA-20 (103 days vs 74.5 days, p=0.014) and NB1691 xenografts. CONCLUSION: In a xenograft model, increased OPG expression correlated with a delay to pathologic fracture suggesting a potential role for mTOR inhibitors in the treatment of neuroblastoma bone metastases.

Rapamycin-induced tumor vasculature remodeling in rhabdomyosarcoma xenografts increases the effectiveness of adjuvant ionizing radiation.

PURPOSE: Rapamycin inhibits vascular endothelial growth factor expression. Vascular endothelial growth factor is a tumor-elaborated protein that stimulates neovascularization. This inhibition can cause transient "normalization" of the generally dysfunctional tumor vasculature, resulting in improved tumor perfusion and oxygenation. We hypothesized that this may potentiate the antitumor effects of adjuvant ionizing radiation. METHODS: Mice bearing orthotopic Rh30 alveolar rhabdomyosarcomas were treated with rapamycin (5 mg/kg intraperitoneally daily ×5). Tumors were then evaluated for changes in intratumoral oxygenation, perfusion, vessel permeability, and microvessel density. Additional tumor-bearing mice were treated with 5 doses of rapamycin, irradiation (4 Gy), or 5 doses of rapamycin with irradiation administered on the first or sixth day of rapamycin treatment. RESULTS: Although tumor vessel permeability changed only minimally, microvessel density decreased (3153 ± 932 vs 20,477 ± 3717.9 pixels per high-power field), whereas intratumoral oxygenation increased significantly (0.0385 ± 0.0141 vs 0.0043 ± 0.0023 mm Hg/mm(3)) after 5 doses of rapamycin. Contrast-enhanced ultrasound demonstrated a significantly increased rate of change of signal intensity after 5 days of rapamycin, suggesting improved intratumoral perfusion. Tumor volume 14 days after treatment was smallest in mice treated with the combination of rapamycin given before irradiation. CONCLUSION: Combination therapy with rapamycin given before irradiation to normalize the tumor vasculature, thereby improving tumor oxygenation, increased the sensitivity of alveolar rhabdomyosarcoma xenografts to adjuvant irradiation.

Continuous local delivery of interferon-ß stabilizes tumor vasculature in an orthotopic glioblastoma xenograft resection model.

BACKGROUND: High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-ß delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection. METHODS: Xenografts were established by injecting 3 grade IV glioblastoma cell lines (GBM6-luc, MT330-luc, and SJG2-luc) into the cerebral cortex of nude rats. Tumors underwent subtotal resection, and then had gel foam containing an adeno-associated virus vector encoding either hIFN-ß or green fluorescence protein (control) placed in the resection cavity. The primary endpoint was stabilization of tumor vasculature, as evidenced by CD34, α-SMA, and CA IX staining. Overall survival was a secondary endpoint. RESULTS: hIFN-ß treatment altered the tumor vasculature of GBM6-luc and SJG2-luc xenografts, decreasing the density of endothelial cells, stabilizing vessels with pericytes, and decreasing tumor hypoxia. The mean survival for rats with these neoplasms was not improved, however. In rats with MT330-luc xenografts, hIFN-ß resulted in tumor regression with a 6-month survival of 55% (INF-ß group) and 9% (control group). CONCLUSION: The use of AAV hIFN-ß in our orthotopic model of glioblastoma resection stabilized tumor vasculature and improved survival in rats with MT330 xenografts.

AAV-mediated local delivery of interferon-beta for the treatment of retinoblastoma in preclinical models.

Interferon-beta (IFN-beta) has been found to have anti-tumor properties against a variety of malignancies through different mechanisms. However, clinical trials involving systemic administration of IFN-beta have been hampered by secondary toxicity and the short half-life of IFN-beta in the circulation. In order to circumvent these limitations, we have developed an adeno-associated viral (AAV) vector gene-therapy approach to deliver IFN-beta to tumors. In this study, we tested the efficacy of AAV-mediated local delivery of IFN-beta for the treatment of retinoblastoma in preclinical models. Retinoblastoma is an ideal candidate for gene-therapy-based anti-cancer treatment because target cell transduction and, therefore, IFN-beta delivery can be contained within the ocular environment, thereby minimizing systemic toxicity. We report here that retinoblastoma cell lines exhibit pleiotropic responses to IFN-beta consistent with previous studies on a variety of tumor cell lines. Intravitreal injection of AAV-IFN-beta resulted in efficient retinal infection and sustained IFN-beta production in the eye with minimal systemic exposure. Vector spread outside of the eye was not detected. Using our orthotopic xenograft model of retinoblastoma, we found that intravitreal injection of AAV-IFN-beta had a potent anti-tumor effect in vivo. These data suggest that AAV-mediated delivery of IFN-beta may provide a complementary approach to systemic chemotherapy which is the standard of care for retinoblastoma around the world.

Bevacizumab suppresses neuroblastoma progression in the setting of minimal disease.

BACKGROUND: We hypothesized that vascular endothelial growth factor (VEGF) contributes to autocrine stimulation of neuroblastoma and that inhibition of its signaling pathway contributes to the anticancer activity of bevacizumab, an anti-VEGF monoclonal antibody. METHODS: For in vitro studies, 2 neuroblastoma cell lines, CHLA-255 and NB1691, were treated with VEGF+/-bevacizumab. For in vivo studies, disseminated neuroblastoma was established by intravenous administration of luciferase-expressing tumor cells in SCID mice prior to bevacizumab treatment. RESULTS: Exogenous VEGF increased cell counts after 48 h (NB1691: 58,878 +/- 8279 vs 137,500 +/- 13,108 cells, P < .001; CHLA: 1.56 x 10(6) +/- 866 vs 1.81 x 10(6) +/- 2550 cells, P <.001); the addition of bevacizumab abrogated this stimulation. In vivo, mice with disseminated disease treated twice weekly with intraperitoneal bevacizumab had a decreased tumor burden at day 14 and prolonged survival (NB1691: 50 +/- 2 vs 43 +/- 2 days, P < .001; CHLA: 53 +/- 3 vs 42 +/- 1 days, P = .006). Interestingly, VEGF and basic fibroblast growth factor expression was increased in treated NB1691 tumors, which likely occurred in response to VEGF signaling inhibition. CONCLUSION: Our results suggest that VEGF has a role in neuroblastoma autocrine signaling. Maintenance therapy with bevacizumab may be useful for disease suppression after maximal cytoreductive therapy; however, upregulation of proangiogenic factors may provide resistance to this approach, which suggests that maximal antitumor efficacy may require combination therapy.

Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates.

The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1x10(12) vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1+/-0.5 microg/mL, or 22% of normal) that were comparable to those achieved after direct delivery of the same vector dose into the portal circulation (1.3+/-0.3 microg/mL, or 26% of normal). Importantly, the pattern of vector biodistribution after systemic and portal vein administration of scAAV-LP1-hFIXco was almost identical. Additionally, comparable levels of gene transfer were achieved in macaques with preexisting immunity to AAV8 following peripheral vein administration of 1x10(12) vg/kg AAV5-pseudotyped scAAV-LP1-hFIXco. This confirms that alternative serotypes can circumvent preexisting naturally acquired immunity to AAV. Thus, peripheral venous administration of AAV5 and AAV8 vectors is safe and as effective at transducing the liver in nonhuman primates as direct vector administration into the portal circulation. These results should make vector administration to patients, especially those with a severe bleeding diathesis, significantly easier and safer.

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 x 10(10) scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.

Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models.

A detailed comparison of recombinant adeno-associated viral (rAAV) vectors of serotypes 2, 5, and 8 was performed in mice and nonhuman primates. Differences within the capsid proteins and viral terminal repeats of rAAV-2 and -5 did not significantly influence their ability to transduce murine liver. However, vectors pseudotyped with AAV-8 capsid (rAAV-2/8) mediated transgene expression more rapidly and from lower doses than possible with rAAV-2 and -5, although expression declined from peak values in a distinct dose-dependent manner prior to reaching steady-state levels. Nevertheless, at all time points and vector doses, rAAV-2/8 transgene levels were 17- to 84-fold higher than with rAAV-2 or -5 due to a more rapid conversion of the single-stranded genome to transcriptionally active stable duplex DNA. In nonhuman primates, liver-targeted administration of rAAV-5 and rAAV-2/8 vectors established therapeutic levels of transgene expression. The importance of preexisting serotype immunity was highlighted by the inability to achieve successful transduction in the presence of serotype-specific antibodies, although this impediment was successfully avoided through the use of alternative serotypes. In summary, serotype-specific differences in transduction biology and the appreciation of preexisting immunity will likely influence the selection of the rAAV serotype for future clinical trials.

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.