Increased Transgene Expression Research Articles

Intrathecal Injection of Naked Plasmid DNA Provides Long-term Expression of Secreted Proteins

Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.

Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3' end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

The effect of the nonionic block copolymer pluronic P85 on gene expression in mouse muscle and antigen-presenting cells

DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO 26–PO 40–EO 26. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c + (DC), and local expansion of CD11c +, CD14 + (macrophages) and CD49b + (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c + CD86 +, CD11c + CD80 +, and CD11c + CD40 +). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.

Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes

Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.

Functional characterization of a tobacco matrix attachment region-mediated enhancement of transgene expression

TM2, a new matrix attachment region (MAR) isolated from tobacco, increases transgene expression in plants. We have carried out a more detailed analysis of the DNA elements in TM2 with the aim of improving its effect on transcription activation. Our study of the location effect of individual MARs on the expression of the adjacent 35S:gusA cassette indicated that the TM2 functions in a bidirectional manner, with the 5'-MAR being more efficient in enhancing beta-glucuronidase expression than the 3'-MAR. The influence of 5'-MAR on different linked mini-promoters in transgenic tobacco cells suggested that the role of TM2 depends on the basic expression of the transgenes. Deletion analysis of one topo II site and two unwinding sites together with one T-box revealed that all these sites contribute most (93.3%) of the transcription activation mediated from the TM2 sequence. Additionally, micrococcal nuclease accessibility of the 35S promoter region can be strengthened by linked TM2, suggesting that the TM2 mediates the spreading of nucleosome opening. Taken together, our results reveal that the TM2 mediates a more open and accessible chromatin DNA structure for promoter-dependent active transcription, which in turn enhances transgene expression.

Complexation of retroviruses with polymers significantly increases the number of genes transferred to murine embryonic stem cells, but does not raise transgene expression levels

Embryonic stem cells efficiently silence retrovirus transgene expression. To help to solve this problem, retroviruses have been developed that are more resistant to silencing, such as retroviruses derived from the MSCV (murine-stem-cell virus). A complementary approach to increasing transgene expression might be to increase the number of integrated transgenes. To test this approach, we formed polymer complexes with MSCV-derived ecotropic retroviruses, concentrated them up to 40-fold and transduced two different murine embryonic stem cell lines, with a mouse fibroblast cell line as a control. The number of integrated transgenes increased more than 50-fold in the embryonic stem cell lines, yet, surprisingly, transgene expression did not increase. Interestingly, the embryonic stem cells had significantly fewer integrated transgenes than the mouse fibroblasts, even though transduction conditions were identical, which suggests that embryonic stem cells may restrict a post-binding step of retrovirus transduction.

Abstract 300: Pathophysiologically Regulated Gene Expression in Human Stem & Progenitor Cells for Cardiogenic Differentiation and Repair

Human stem and progenitor cells have emerged as potentially useful substrates for cardiovascular repair through neovascularization and myocardial regeneration. However, efficacy is limited by impedance to stem cell retention, homing and differentiation in hostile microenvironments, as occur in infarcted myocardium. The objective of the current study was to regulate gene function for tailored therapy in post infarct myocardium. Here we show that hypoxic and inflammatory stimuli of the infarct microenvironment regulate a proportional response in gene expression in human endothelial progenitor (EPC) and mesenchymal stem cells (MSC). Highly efficient lentiviral vectors incorporating hypoxia (HRE) and nuclear factor kappa B (NFkB) responsive elements are used to drive transgenes for survival, autologous stem cell homing and cardiogenic differentiation. Utilizing an internal cytomegalovirus promoter deleted lentiviral transfer vector, an HRE-NFkB bicistronic promoter-reporter vector was constructed with a modified internal ribosome entry sequence between green fluorescent protein and luciferase or therapeutic genes. Either hypoxia or inflammation resulted in a seven to ten-fold response of transgene expression assessed by luciferase activity in EPC (hypoxia, 7608±954; inflammation 11492±1384, P<0.01 and P<0.001 vs control 1049±139 respectively, N=6), while combined hypoxic-inflammatory stimuli resulted in a sixty-fold increase of transgene expression (hypoxic-inflammation, 62364±6609, P<0.001 vs control 1049±139, N=6). These results were recapitulated in MSC and with a series of therapeutic genes as determined by transcript, protein expression and activity. Our results demonstrate that regulated vectors provide a proportional response to hostile post-infarct myocardium. Translating cardiovascular regenerative medicine using stem cells requires managing stem cell survival, function and differentiation. Utilizing site-specific pathophysiological cues to auto-regulate reparative and regenerative gene expression, this study is a starting point for sophisticated platforms for patient tailored cell-based cardiogenic therapy.

Sustained Transgene Expression Using MAR Elements

Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.

Substitution of Hexon Hypervariable Region 5 of Adenovirus Serotype 5 Abrogates Blood Factor Binding and Limits Gene Transfer to Liver

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Effects of Inflammatory Response on In Vivo Transgene Expression by Plasmid DNA in Mice

To examine the effects of inflammatory response to plasmid DNA (pDNA) on transgene expression, serum tumor necrosis factor-alpha (TNF-alpha) was measured after intravenous injection of pDNA or calf thymus DNA (CT DNA) in the naked or complexed form with cationic liposomes (lipoplex). pDNA with many CpG motifs induced TNF-alpha production regardless of the forms. No significant TNF-alpha production was detected when CT DNA or methylated pDNA was injected. Clodronate liposomes and dexamethasone were used to deplete phagocytes or to inhibit inflammatory responses, respectively. Transient depletion of phagocytes, such as liver Kupffer cells and splenic macrophages, by clodronate liposomes slightly altered the tissue distribution of (32)P-pDNA lipoplex, but significantly reduced the TNF-alpha production and transgene expression. Dexamethasone significantly inhibited the initial transgene expression, but increased the duration of the expression slightly. Use of NF-kappaB activity-dependent plasmid vector suggested that the inhibition of NF-kappaB activation is involved in the reduced expression by these treatments. These findings indicate that tissue macrophages are closely involved in the CpG motif-dependent TNF-alpha production. It is also suggested that TNF-alpha activates NF-kappaB and increases transgene expression by pDNA having many NF-kappaB binding sites, but TNF-alpha also reduces transgene expression at later time periods, leading to short-term transgene expression.

Macrophage Depletion Combined with Anticoagulant Therapy Increases Therapeutic Window of Systemic Treatment with Oncolytic Adenovirus

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad "detargeting" from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor-dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites.

Effects of mutant human Ki- rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki- ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki- ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki- ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.

Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles

Endosomal release is an efficiency-limiting step for many nonviral gene delivery vehicles. In this work, nonviral gene delivery vehicles were modified with a membrane-lytic peptide taken from the endodomain of HIV gp41. Peptide was covalently linked to polyethylenimine (PEI) and the peptide-modified polymer was complexed with DNA. The resulting nanoparticles were shown to have similar physicochemical properties as complexes formed with unmodified PEI. The gp41-derived peptide demonstrated significant lytic activity both as free peptide and when conjugated to PEI. Significant increases in transgene expression were achieved in HeLa cells when compared to unmodified polyplexes at low polymer to DNA ratios. Additionally, peptide-modified polyplexes mediated significantly enhanced siRNA delivery compared to unmodified polyplexes. Despite increases in transgene expression and siRNA knockdown, there was no increase in internalization or binding of modified carriers as determined by flow cytometry. The hypothesis that the gp41-derived peptide increases the endosomal escape of vehicles is supported by confocal microscopy imaging of DNA distributions in transfected cells. This work demonstrates the use of a lytic peptide for improved trafficking of nonviral gene delivery vehicles.

A fusogenic segment of glycoprotein H from herpes simplex virus enhances transfection efficiency of cationic liposomes

Low cellular uptake and endosomal escape are regarded as major limitations for nonviral gene delivery. In the present study, we use cationic liposomes incorporating fusogenic peptides from glycoprotein H of herpes simplex virus to improve cellular internalization and endosomal release of cationic liposome-DNA complexes. A synthetic analogue of a fusogenic peptide domain in glycoprotein H from herpes simplex virus was evaluated for in vitro gene delivery. The fusogenicity of the peptide was evaluated by a lipid mixing assay in neutral and acidic environments. The influence of the peptide on cellular internalization and subcellular distribution of Lipofectamine-pGL3 complexes were evaluated by flow cytometry, fluorescence microscopy, and confocal laser scanning microscopy. The effect of the peptide on transfection efficiency of cationic liposomes was also evaluated by luciferase assay in human cell lines. A pH-sensitive fusogenicity of the wild-type peptide was observed. In the lipid mixing assay, the peptide mediated 80% membrane fusion at pH 7.4 at a 0.05 : 1 (peptide : lipid) mole ratio. At pH 4.5, 100% of membrane fusion was observed at a 0.005 : 1 ratio. Adding the peptide to Lipofectamine-pGL3 complexes significantly increased cellular uptake and, thus, increased transgene expression up to 30-fold in human cell lines. Nuclear localization of the DNA complex by the fusogenic peptide was observed by confocal microscopy. A synthetic analogue of a fusogenic peptide domain from herpes simplex virus has been proven to improve cationic lipid-mediated transfection in vitro. The application of this fusogenic peptide will lead to improved strategies for transfection and successful gene therapy.

Chicken β‐globin insulator overcomes variegation of transgenes inXenopusembryos

Chromatin structure and gene transcription regulation are intimately linked, and mosaic expression of randomly integrated transgenes into the genome is frequently observed. This variegation of transgene expression is likely due to the genomic integration site, which can affect the behavior of the integrated DNA sequence in a positive or a negative way. Insulators are a class of DNA elements that can protect genes from inappropriate signals emanating from their environment by acting as boundaries that prevent the spreading of nearby condensed chromatin that may otherwise silence expression. Here we show that transgenes escape this silencing in Xenopus laevis and Xenopus tropicalis embryos and that a stable, uniform, and heritable expression pattern is obtained when transgenes are flanked with tandem copies of the chicken beta-globin 5'HS4 insulator. Our data also indicate that the insulator confers copy-number-dependent transgene expression and can increase transgene expression from weak regulatory elements. Hence, it will be an invaluable tool for generating stable lines expressing different levels of a particular coding sequence.

A simple, lanthanide-based method to enhance the transduction efficiency of adenovirus vectors

Based upon the powerful bridging and charge-masking properties of lanthanide cations (Ln3+), we have investigated their use to improve the transduction efficiency of adenovirus vectors. Using a luciferase marker gene, it was possible to increase transgene expression by the murine mesenchymal stem cell line C3H10T(1/2) by up to four log orders when using very low multiplicities of infection in conjunction with Ln3+; La3+ was superior to Gd3+, Y3+ and Lu3+ in this regard. All Ln3+ were more effective than Ca2+. Flow cytometry, using a green fluorescent protein marker gene, confirmed that La3+ increased both the percentage of transduced cells and the level of transgene expression per cell. Transduction of primary cultures of a variety of different mesenchymal cells from human, rabbit, bovine and rat sources, as well as gene transfer to synovium and muscle in vivo, was also greatly enhanced. Our findings suggest that this lanthanide-based method holds much promise for expediting both experimental and clinical applications of gene transfer with adenoviral vectors.

Augmented transgene expression in transformed cells using a parvoviral hybrid vector

Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.

RAAV vector-mediated gene therapy for experimental ischemic stroke

The safest viral vector system for gene therapy is based on recombinant adeno-associated virus (rAAV) up to date in Phase I clinical trials, which has been developed rapidly and applied for ischemic stroke gene therapy in animal experiments since the past seven years. rAAV vector has made great progress in improving gene delivery by modification of the capsid and increasing transgene expression by encapsidation of double-stranded rAAV genome. And in all, nine therapeutic genes in 12 animal studies were successfully delivered using rAAV vector to ischemic brain via different approaches in rat or mice stroke models for gene therapy and the results suggested that rAAV could mediate genes' expression efficiently; most of them displayed evidently therapeutic efficacy with satisfactory biological safety. Gene therapy involving rAAV vector seems effective in attenuation of ischemic damage in stroke and has greatly promising potential use for patients in the future. In this review, we will focus on the basic biology and development of rAAV vector itself as well as the recent progress in the use of this vector for ischemic stroke gene therapy in animal experiments.

Differential Effects of Combined Ad5-Δ24RGD and Radiation Therapy in In vitro versus In vivo Models of Malignant Glioma

The integrin-targeted conditionally replicating adenovirus Ad5-delta 24RGD has been shown to possess strong oncolytic activity in experimental tumors and is currently being developed toward phase I clinical evaluation for ovarian cancer and malignant glioma. Previously, we reported that combination therapy of Ad5-delta 24RGD with irradiation led to synergistic antitumor activity in s.c. glioma xenografts. In the current study, the underlying mechanism of action to this synergy was studied and the effects of combined therapy were assessed in an orthotopic glioma model. Sequencing studies in U-87 monolayers showed that delivery of irradiation before Ad5-delta 24RGD infection led to a greater oncolytic effect than simultaneous delivery or infection before irradiation. This effect was not due to enhanced virus production or release. Experiments using a luciferase-encoding vector revealed a small increase in transgene expression in irradiated cells. In tumor spheroids, combination therapy was more effective than Ad5-delta 24RGD or irradiation alone. Staining of spheroid sections showed improved penetration of virus to the core of irradiated spheroids. Mice bearing intracranial tumors received a combination of Ad5-delta 24RGD with 1 x 5 Gy total body irradiation or with 2 x 6 Gy whole brain irradiation. In contrast to the in vitro data and reported results in s.c. tumors, addition of radiotherapy did not significantly enhance the antitumor effect of Ad5-delta 24RGD. Combined treatment with Ad5-delta 24RGD and irradiation shows enhanced antitumor activity in vitro and in s.c. tumors, but not in an orthotopic glioma model. These differential results underscore the significance of the selected tumor model in assessing the effects of combination therapies with oncolytic adenoviruses.

Safety of Recombinant Adeno-Associated Viral Vectors in a Large Animal Model.

Recombinant adeno-associated viral (AAV) vector is a promising gene-based strategy for the treatment of several inherited diseases. Using AAV serotype 2 (AAV-2), the most common tested vector in humans, we have determined that the risk of germline transmission and the immune responses to both transgene product and/or vector-capsid proteins are critical obstacles to the safety of this strategy (Nat Med 12:342, 2006). Recently, novel and more potent serotypes have emerged such as AAV-8 that allows efficient liver transduction following peripheral intravenous injection (IV). The major determinant of vector safety relies on its tissue tropism. Here, we sought to compare the efficacy and safety profile of AAV-2 and AAV-8 in a large animal model. Male rabbits (∼3 kg) received IV injection of AAV-2 (n=11) or AAV-8 (n=11) encoding human F.IX (hF.IX) at doses ranging from 1×1012 to 1×1013vg/kg. Injections with AAV-2-hF.IX resulted in 6-fold lower expression of hF.IX than AAV-8-hF.IX for both low and high dose cohorts. Notably, no neutralizing antibody to hF.IX was detected with either serotypes. Eighteen weeks following the initial injections, animals were cross-administered with either AAV-2 or AAV-8. Whereas injection of AAV-8 led a 20% increase in transgene expression in animals initially injected with AAV-2-h.FIX, AAV-2 failed to boost hF.IX expression. Regarding germline safety, the presence of vector genomes in semen samples from the high-dose cohort (6 to 10 weeks after injection) was 3-5 fold higher for AAV-8 compared with AAV-2. There were no differences in the vector clearance in the semen among rabbits from the low-dose cohorts of AAV-2 and AAV-8. After 12 weeks, all semen samples from all cohorts tested negative. In another rabbit model, vasectomized prior to vector injection, we determined that semen samples lacking germ cells were also positive for vector-DNA sequences. The kinetics of vector clearance in these samples was dose- and time-dependent and serotype-independent. Because the presence of capsid in early-time points is critical for predicting possible immune responses against the viral vector, we determine the vector biodistribution one week after injection of 1×1013 vg/kg of AAV-2 or AAV-8. Rabbits were euthanized and their organs were harvested and analyzed for vector DNA presence through real-time quantitative PCR. Comparing to AAV-2, AAV-8 genomes were 2-5 fold times higher in all organs (spleen, lung, heart, and kidney), with the exception of liver where vector-DNA content was comparable (range from 25-69 copy number/cell). In addition, testes, accessory glands, and prostates were positive for the vector DNA, albeit in very low levels (the highest level of vector DNA found in those organs was 3copy number/cell in the testis of one rabbit injected with AAV-8). These differences may reflect the distributions of cellular receptors for AAV-2 and AAV-8, which may also explain the higher content of vector genome in the semen of high-dose AAV-8 cohort. Together our findings suggest that AAV-8 ensures higher transgene expression than AAV-2 and preexisting immunity to AAV-2, a naturally acquired virus in humans, may not limit AAV-8-mediated gene delivery. The overall kinetics of AAV vector clearance in the semen seems to be independent of the presence of germ cells and vector serotype. However, early biodistribution data of AAV-8 suggests a distinct safety profile from AAV-2.