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Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Although many types of drug are used, clinical outcomes are still unsatisfactory. Previous studies have suggested that intestinal bacteria are involved in the pathogenesis of IBD. Accordingly, in an IBD model we evaluated the therapeutic effects of OPS-2071, a low-absorption quinolone antibacterial agent indicated for intestinal infection, and investigated its mechanism of action. The therapeutic effects of OPS-2071 and comparison therapies were evaluated using naive CD4 + T cell-transfer IBD model mice. In vitro inhibition of LPS-induced TNF-α production and inhibitory effects on T cell responses stimulated using anti-CD3/CD28 antibody-loaded beads were evaluated using mouse splenocytes and human peripheral blood mononuclear cells. In addition, in vitro activities against bacteria implicated in IBD pathogenesis were tested. OPS-2071 dose-dependently decreased both colonic weight/length ratio and the colitis histological score as compared with the vehicle group. The therapeutic effect of OPS-2071 was equivalent to that of anti-IL-12/23 (p40) antibody. In vitro, OPS-2071 suppressed TNF-α production induced by LPS stimulation and T cell responses in a dose-dependent manner. At high concentrations, these effects were comparable to those of existing immunosuppressive agents, such as prednisolone, in both mouse and human cells. OPS-2071 also showed antibacterial activity against IBD-related bacteria. Our results suggest that OPS-2071 had both immunosuppressive and antibacterial effects. This dual effect makes OPS-2071 a unique and promising candidate for IBD.

OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin., OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin. OPS-2071 exhibited potent antibacterial activity against 54 clinically isolated C. difficile strains with a MIC of 0.125 μg/ml (MIC50) and 0.5 μg/ml (MIC90), making it more active than vancomycin on a concentration basis (MIC50, 2 μg/ml; MIC90, 4 μg/ml) and comparable to fidaxomicin (MIC50, 0.063 μg/ml; MIC90, 8 μg/ml). OPS-2071 showed equally potent antibacterial activity against both hypervirulent and nonhypervirulent strains, while a significant difference in susceptibility to fidaxomicin was observed. Spontaneous resistance to OPS-2071 and vancomycin was not observed; however, resistance to fidaxomicin was observed at 4× MIC. The mutant prevention concentration of OPS-2071 was 16-fold lower than those of fidaxomicin and vancomycin, and the postantibiotic effect of OPS-2071 was longer than those of fidaxomicin and vancomycin. Also, OPS-2071 showed low systemic exposure, with OPS-2071 having 2.9% oral bioavailability at 1 mg/kg in rats. Furthermore, OPS-2071 showed significant in vivo efficacy at 0.0313 mg/kg/day (50% effective doses), 39.0-fold and 52.1-fold lower than those of vancomycin and fidaxomicin, respectively, in a hamster model of C. difficile infection. OPS-2071 has the potential to become a new therapeutic option for treating C. difficile infection.

Background Crohn’s disease (CD) is a form of chronic inflammatory bowel disease (IBD). Although CD is an immune-mediated condition of unknown etiology, many studies suggested that abnormal immune responses against certain intestinal bacteria may trigger development of chronic inflammation. The T cell-mediated colitis model is a well characterized adaptive transfer murine model of chronic small bowel and colonic inflammation that resembles human CD (e.g., diarrhea, a heavily inflamed colon, loss of mucus from goblet cells, Th1/Th17 dominated cytokine profile). OPS-2071, a novel agent synthesized by Otsuka Pharmaceutical Co., Ltd, has broad and strong antibacterial activity with low systemic absorption. In this study, we have evaluated the therapeutic effect of OPS-2071 on murine T cell-mediated colitis and in vitro activity on T cell activation, cytokine production and antibacterial activity. Methods T cell-mediated colitis was induced via naive T cell (CD4+CD62L+CD44-) injection into T and B cell deficient recipient mice. Two weeks following T cell transfer, OPS-2071 or vehicle solution was administered orally for 3 weeks, followed by harvesting of colon tissue and assessment of efficacy evaluation by histological score and inflammatory index (colon weight/ length). In vitro inhibitory action for TNF-α production was assayed by ELISA using LPS-stimulated human peripheral blood cells. The effect on T cell activation and cytokine production (TNF-α, IFN-γ), was examined using human peripheral blood mononuclear cells and, mouse spleen which were stimulated by anti-CD2/CD3/CD28 antibody-loaded beads. In vitro activity against bacteria considered to be the cause of IBD was also tested. Results In the murine T cell transfer model, OPS-2071 significantly reduced both histological score (control: 9.2, OPS: 2.7, p128 mg/mL, respectively. Conclusion OPS-2071 demonstrated significant therapeutic effects on colon inflammation in the murine T cell-medicated colitis model, suppressed TNF-a production in vitro and showed in vitro activity against bacteria related to CD. The dual effect of anti-inflammatory effects and antibacterial activity of the novel agent OPS-2071 demonstrated in this study, provide rationale for exploring the impact of this compound on human CD in clinical trials.

Background OPS-2071 is a novel fluoroquinolone that is currently in Phase 2 clinical trials for the evaluation of Crohn’s disease (CD) as an add-on therapy to standard of care. OPS-2071 has broad and potent antibacterial activity with low systemic absorption. It was previously investigated for the treatment of enteric infections, including Clostridium difficile. CD is an immune-mediated condition of unknown aetiology; however, studies suggest that an abnormal immune response against certain intestinal bacteria may trigger the development of chronic inflammation. In this study, we have evaluated the effect of OPS-2071 on both the murine T-cell-mediated colitis as well as its in vitro effect on the activity of immune cells. Methods Transfer of naïve T cells (CD4+CD62L+CD44−) into immunodeficiency mice induces autoimmune colitis. Two weeks following T-cell transfer, OPS-2071, sulfasalazine and anti-IL12/23 p40 antibody were administered for 3 weeks, followed by assessment of efficacy evaluation by histological score and inflammatory index. In vitro inhibitory action for TNF-α production was assayed using LPS-stimulated human peripheral blood cells and THP-1 macrophages. The effect on T-cell activation and cytokine production (TNF-α, IFN-γ) was examined using anti-CD2/CD3/CD28 antibody-loaded beads-stimulated human peripheral blood mononuclear cells. In vitro activity against bacteria considered to be a potential cause of IBD such as M. avium sub. paratuberculosis, Klebsiella pneumonia, etc., was also tested. Results OPS-2071 and anti-IL12/23 p40 antibody significantly reduced both histological score (control: 9.0, OPS: 4.7, anti-IL12: 4.3, p < 0.01) and inflammatory index (control: 58.3 ± 3.7 mg/cm, OPS: 25.9 ± 1.4 mg/cm, anti-IL12: 41.1 ± 2.4 mg/cm, p < 0.01) at a dose of 10 and 25 mg/kg, respectively. OPS-2071 suppressed TNF-α production in T cells, as well as macrophage activity in a dose-dependent fashion. OPS-2071 also suppressed human T-cell activation and proliferation. At high OPS-2071 concentrations, these effects were comparable to prednisolone. The minimum inhibitory concentration against IBD-related bacteria for: OPS-2071, ciprofloxacin, and metronidazole were 0.015–0.5, 0.25–8 and 0.03–>128 mg/ml, respectively. Conclusion OPS-2071 demonstrated significant therapeutic effect on colonic inflammation, suppressed TNF-α production in vitro and showed in vitro activity against bacteria related to CD. From these in vitro results, it can be concluded that OPS-2071 has anti-inflammatory activity, independent of its antibacterial activity. The dual effects demonstrated by this novel agent, OPS-2071, provided a rationale for exploring the impact of this compound on human CD in clinical trials.

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.

In order to study the production system of polyunsaturated fatty acids (PUFA), we genetically engineered a baker’s yeast that did not originally produce PUFA. We produced γ-linolenic acid (GLA) by constructing a heterologous system to express Δ6 fatty acid desaturase (D6d) gene from rat liver into yeast, Saccharomyces cerevisiae. The expression of this gene in the presence of linoleic acid (LA) formed a significant amount of GLA in the host cells. In order to effect the largest expression of the conversion rate from LA to GLA, we set up a dual promoter system, where genes coding for the D6d and an elecron donor protein, cytochrome b5, were coordinately expressed. The desaturation index (GLA/LA) increased significantly by high level expression of cytochrome b5, indicating that this protein is a limiting factor in regular yeast. In addition, we examined a condition for the extra-cellular production of GLA by a fatty acid secretion yeast mutant. By expressing the D6d gene under the control of GAP-DH promoter in the presence of LA, 178 mg/L of GLA was secreted in the medium in 144h.

Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations. These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone. Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid. In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate. These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast.

Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 × 10 11 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a β-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ∼0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was ∼0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10 3 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.

Adeno-associated virus (AAV) is a small nonpathogenic, replication-defective DNA virus with a single-stranded DNA genome. Recombinant AAV (rAAV) is among the most promising gene delivery vectors for human gene therapy (15, 28). rAAV lacks machinery for virus genome insertion into host chromosomal DNA but does integrate into chromosomes at a low frequency (reviewed in reference 29). Murine leukemia virus preferentially integrates near transcription start sites (59), while human immunodeficiency virus favors transcribed regions for integration (49). As for rAAV, previous studies have shown that rAAV serotype 2 (rAAV2) vector integration occurs preferentially in genes and near gene regulatory sequences in mouse liver (36, 40) and in cultured human cells (32). A recent observation by Miller et al. has suggested that rAAV does not cause chromosomal breaks but integrates at preexisting chromosomal breakage sites (30). Although rAAV vector integration frequency is considered to be low, it is important to further understand the interactions between rAAV vector and host chromosomal DNA in various tissues in experimental animals. This is because a study has demonstrated increased incidence of liver cancer in rAAV2 vector-treated animals (5), the mechanisms for which remain elusive. In addition, the use of new serotype vectors with robust transduction efficiency, such as rAAV8, can increase vector genome loads in cells, which may pose an increased risk of undesirable genomic alterations in rAAV-transduced cells. Such robust serotype vectors now are widely used for many preclinical studies for gene therapy of various human diseases. Moreover, we have hypothesized that elucidation of the mechanisms of rAAV integration may help in understanding ongoing genomic instability in living animals, which is difficult to investigate with currently available strategies but is important for studies on carcinogenesis and aging. Furthermore, it has been shown that rAAV vectors serve as a powerful tool to study the mechanisms of fundamental biological processes in cells, such as DNA damage responses and DNA repair (11). To begin to further understand rAAV integration in various tissues of experimental animals, it is essential to establish a system by which rAAV integration sites can be identified on a large scale in nondividing cells with high efficiency and reliability without any cell manipulations. This minimizes possible technical biases and enables identification of rAAV integrations in nonhepatic tissues, for which selection is not easy to perform. However, currently available methods for high-throughput rAAV integration site analysis all rely on cell division either under a selective pressure (40) or without selection (32). Cell division is required for diluting out extrachromosomal rAAV vector genomes with high complexity, which are abundantly present in cells in a quiescent state and inhibit efficient isolation of rAAV integrants. In the present study, we have established a novel high-throughput method to identify rAAV integration sites in nondividing cells with high efficiency and high reliability independently of cell division, transgene expression, or selection. We have identified a thousand rAAV integration sites in quiescent somatic cells in mouse liver, skeletal muscle, and heart, and we discovered that DNA palindromes are a common target for rAAV integration. DNA palindromes are found prevalently in many organisms, including mammals (24, 58; S. M. Lewis, T. Zheng, S. Chen, T. Alleyne, J. Cheung, T. Chiang, and R. Richard, unpublished data). They have gained attention recently due to accumulating evidence that they have roles in promoting genomic instability in eukaryotes. This DNA motif has been shown to be involved in gene amplification (2, 7, 43, 46, 52-54, 60), nonrandom chromosomal translocations causing human diseases (6, 9, 12, 16-20, 55), genomic instability in animal models (1, 3, 4, 22, 23), retrovirus integration (14), and RAG protein-mediated transposition (45). Thus, the discovery that rAAV integrates preferentially at DNA palindromes not only provides further insights into the mechanisms of rAAV integration but also provides an unprecedented opportunity to study the biological impact and properties of naturally occurring DNA palindromes in tissues of living animals.

We previously demonstrated that major membrane protein II (MMP-II) is one of the immunodominant antigens (Ags) ofMycobacterium lepraecapable of activating T cells through Toll-like receptor 2. Based on the observation thatMycobacterium bovisBCG secreting a 30-kDa protein offered better protection against tuberculosis, we constructed a recombinant BCG strain (BCG-SM) that secretes MMP-II to improve the potency of BCG against leprosy. The secreted MMP-II protein from BCG-SM stimulated monocyte-derived dendritic cells (DC) to produce interleukin-12. DC infected with BCG-SM expressed MMP-II on their surfaces, and MMP-II expression was suppressed by the pretreatment of DC with chloroquine. These results indicated that secreted MMP-II was processed by DC for higher expression levels on their surfaces. In addition, BCG-SM phenotypically activated DC and induced higher expression levels of major histocompatibility complex, CD86, and CD83 Ags on DC than did vector control BCG (BCG-pMV). The DC infected with BCG-SM more efficiently stimulated naïve and memory CD4+T cells and memory CD8+T cells to produce gamma interferon than did those infected with BCG-pMV. However, naïve CD8+T cells were significantly activated only when they were stimulated with BCG-SM-infected DC. When CD8+T cells were cocultured with BCG-SM-infected DC, the proportion of perforin-producing T cells was significantly higher than that in cells cocultured with BCG-pMV-infected DC. Moreover, MMP-II-specific memory T cells were more efficiently produced in mice inoculated with BCG-SM than in mice inoculated with BCG-pMV. Taken together, these results indicate that BCG capable of secreting the immunodominant Ag is more potent in the stimulation of T cells.

rAAV8 vectors transduce various tissues with extremely high efficiency in neonates and infants. Many applications of rAAV8 vector injection into prenatal or neonatal mice have been proposed to treat various disorders. Intraperitoneal or intravascular injection of rAAV8 vector into neonatal mice results in widespread efficient transduction in heart and skeletal muscles, which can stably persist even at adult ages. In contrast, the liver is transduced efficiently with rAAV8 only in the neonatal period, and liver transduction substantially declines thereafter. Despite the limited efficacy in neonatal liver transduction, it is important to understand the biology of rAAV8 vectors in the liver following neonatal administration because 1) the neonatal liver is an ideal target organ for gene transfer in many instances; and 2) substantial vector genome dissemination to the liver is likely to occur in non-hepatic gene transfer. In order to substantially understand the biology of rAAV8 injected into neonates, we non-injected or injected intraperitoneally C57BL/6 and 44Bri mice with AAV8-EF1|[alpha]|-nlslacZ vector at birth, and followed up total 69 animals. 44Bri is an HBV transgenic strain that develops chronic liver injury at |[thksim]|3 months of age, causing continuous liver regeneration. At the age of 110|[ndash]|179 days, the mice received AAV9-LSP (liver-specific promoter)-hFIX vector via the tail vein. 17 days post rAAV9 injection, half of the mice underwent 2/3 partial hepatectomy (PHx), and all the mice were sacrificed 6 weeks after PHx. The rAAV9 vector served as an internal control for PHx in rAAV8-injected animals. Xgal staining of the liver sections revealed sparsely scattered single blue hepatocytes and occasional clusters comprising 2|[ndash]|20 blue hepatocytes. Non-parenchymal blue cell foci were also found in many liver sections analyzed. Several relatively large blue hepatocyte clusters comprising of supposedly 10e2|[sim]|10e4 hepatocytes were found in one wild type and two 44Bri mice after PHx, suggesting that rAAV8-transduced cells in these foci might have had growth advantage over surrounding non-transduced hepatocytes. hFIX expression from the rAAV9 and rAAV9 genome copy numbers in the liver substantially dropped (over 90%) by PHx, while rAAV8 vector copy number drops were significantly smaller than those of rAAV9 vector (27-92% vector copy number drops for wild type and 73% drop-869% increase for 44Bri mice). This indicates that, although a significant portion of rAAV8 vector genomes were extrachromosomal, vector genomes were apparently integrated into chromosomes, with frequency of at most 2.7 vector genome copy/cell in our study. Thus, rAAV8 injected at birth integrates into chromosomes in the liver, stably transducing both parenchymal and non-parenchymal hepatic cells sparsely. In parallel to this experiment, we are following up 69 non-injected and 117 rAAV8-injected (at birth) wild type and 44Bri male mice over 400 days to investigate procarcinogenic potential of rAAV8 in mice. A preliminary result in this study will be also presented.

Top of pageAbstract DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in rAAV vector transduction. Recently, it has been reported that rAAV vector genome integration into chromosomes is substantially increased in scid mouse muscles and livers (PNAS 98: 4084, 2001; PNAS 101:2112, 2004). In scid livers, they observed that >40% of transgene expression and >50% of vector genomes remained after partial hepatectomy (PHx). However, we have not been able to confirm their observations. Rather we have found that liver regeneration is impaired and slowed in scid mice, particularly in older animals, resulting in insufficient dilution of episomal genomes after PHx. In order to revisit this subject, we have established a method that can directly quantify integrated vector genomes by Southern blot, and performed an experiment with a careful monitoring of liver regeneration after PHx. Since we found that animal age is a critical factor for liver regeneration in scid mice, we prepared relatively young age-matched 8-week-old C57BL/6 and C57BL/6 scid male mice. We injected these mice with AAV2 or 8-ISceI.AO3 vector as indicated in Table 1 (n=5|[ndash]|6 per group). The AAV-ISceI.AO3 vectors had an ISceI site in their vector genomes. With these vectors and ISceI digestion, we could clearly separate episomal and integrated vector genomes in transduced tissues, therefore quantify the amount of integrated vector genomes by Southern blot. 7 weeks after injection, 3 mice from each group underwent PHx, and all the mice were sacrificed 8 weeks after PHx. Careful liver weight measurement revealed that liver regeneration in scid mice was impaired by a factor of 0.88. Vector genome copy numbers at and 8 weeks after PHx are summarized in Table 1. The results demonstrate that vector genome copy numbers substantially dropped by 71|[ndash]|90% in scid mouse livers, and there was no significant difference in the reduction of vector genome copy numbers between B6 and scid mice. This sharply contrasts with the previous observations by Song et al. In addition, Southern blot analysis that can quantify integrated vector genomes revealed no detectable integrated vector genomes in any of the liver, skeletal muscle and kidney. Thus we conclude that scid phenotype does not substantially increase rAAV vector genome integration frequency at a detectable level in vivo.

A major part of the palmitic acid (C16:0) generated by fatty acid synthase is converted into stearic acid (C18:0) via carbon chain elongation. Here, we describe the cloning and expression of a rat hepatic enzyme, rELO2, responsible for the elongation of C16:0, presumably at the condensing reaction. Heterologous expression experiments in a yeast, Saccharomyces cerevisiae, demonstrated the elongation activity of rELO2 on C16:0 and to a lesser extent, C18:0 and fatty acids with low desaturation degree. This was distinct from that rELO1, a rat homolog of HELO1, which preferably catalyzed the elongation of mono- and polyunsaturated fatty acids of C16–C20. The Northern analysis showed that the expression of rELO2, but not rELO1, in hepatocytes was activated by the cycles of fasting and refeeding rats on a fat-free diet. Under these conditions, the rELO1 was expressed constitutively in various tissues but the rELO2 transcripts were detected predominantly in liver.

Top of pageAbstract We reported last year that double-stranded (ds) linear rAAV vector genomes with a |[ldquo]|dog bone|[rdquo]| structure (i.e., a dumbbell-shaped ds rAAV genome with both terminal hairpin loops closed) emerged in mouse hepatocytes in the absence of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs, i.e., scid phenotype) but not in the presence of DNA-PKcs (i.e., wild type) when a rAAV8 vector was injected via the tail vein at a very high dose, 7.2 |[times]| 10e12 vector genome (vg) per mouse. We and others have shown that such an altered fate of rAAV vector genomes in murine liver was not observed when rAAV2 vectors were injected at a regular dose. Based on this observation, we have hypothesized that 1) AAV-ITRs are recognized by cells as |[ldquo]|closed|[rdquo]| hairpins whether or not the ITRs have a 5' or 3' DNA end (i.e., |[ldquo]|open|[rdquo]| ITRs as opposed to the |[ldquo]|closed|[rdquo]|), and 2) hairpin opening of |[ldquo]|closed|[rdquo]| ITRs by a specific cellular endonuclease(s) is the very first step that triggers all vector genome recombinations toward the formation of ds circular monomer genomes and concatemers, and vector genome integration into chromosomal DNA in vivo. If AAV-ITR hairpins are not resolved, 5' and 3' DNA ends in vector genomes are ligated with each other intramolecularly by a cellular ligase activity, forming the |[ldquo]|dog bone|[rdquo]| structure, and further intermolecular recombinations through the ITRs would not occur. In the present study, we further investigated the |[ldquo]|dog bone|[rdquo]| genomes as a tool to understand the mechanisms of AAV-ITR hairpin opening and rAAV vector genome recombinations in mice. Consequently, we have elucidated the following: (1) |[ldquo]|dog bone|[rdquo]| genomes emerged in scid liver only when rAAV8 vectors were injected at doses of 1.8 |[times]| 10e12 or higher, at which vector copy numbers in the livers were > 800 ds-vg/dge (ds vector genomes per diploid genomic equivalent); (2) rAAV vectors delivered to scid liver by rAAV2 vectors never resulted in accumulation of |[ldquo]|dog bone|[rdquo]| genomes no matter how much vectors were injected; (3) in non-hepatic scid tissues such as heart, muscle and kidney, |[ldquo]|dog bone|[rdquo]| genomes accumulated at a much lower vector genome load (|[sim]|2 ds-vg/dge); (4) hepatocyte cell cycling recruited a supplementary AAV- ITR hairpin opening activity that did not exist in a quiescent state, facilitating vector genome recombinations through the AAV-ITRs. These observations demonstrate that AAV-ITR hairpin opening in non-hepatic tissues largely depends on the DNA-PKcs-associated cellular endonuclease activity, while in the liver, the pathways of AAV-ITR hairpin opening are redundant and cell cycle dependent. Based on the recent discovery of DNA-PKcs/Artemis pathway for DNA double-strand break repair together with our observations, we propose that a cellular endonuclease, presumably Artemis, plays a crucial role in AAV-ITR hairpin opening and subsequent vector genome recombinations in concert with DNA-PKcs. We are currently investigating the role of Artemis in this process using Artemis deficient cells.

Recombinant AAV(rAAV) vectors are promising vehicles for achieving stable transduction in vivo. We have demonstrated that, in contrast to rAAV2 vectors, which have limited transduction efficiency in many tissues, rAAV8 vectors can transduce all the hepatocytes, cardiomyocytes and skeletal myofibers throughout the body, and a majority of pancreatic acinar cells by peripheral vein injection at a high dose, in mice. AAV serotype 9 has been recently isolated from non-human primate tissues and recombinant vectors based on this serotype have been shown to transduce various tissues with high efficiency. In order to characterize the behavior of rAAV9 vectors in mice, we injected mice with various rAAV9 vectors via several different routes and analyzed transgene expression and tissue distribution. First, we produced a liver specific promoter-driven human factor IX (hFIX)-expressing rAAV9 vector, AAV9-hFIX16, and injected adult C57BL/6 male and female mice via 4 different routes; i.e., portal vein (PV), tail vein (TV), intraperitoneal (IP) and subcutaneous (SC) injections at a dose of 8.0|[times]|10e10 vg/mouse. For comparison, we also injected mice with AAV8-hFIX16 at the same dose. Liver transduction efficiency was monitored by measuring hFIX levels in the plasma. Two weeks post-injection, PV injection with the rAAV9 vector (9PV), 9TV and 9IP achieved comparable hFIX levels of 350-400 |[mu]|g/ml, which was slightly higher than the mice injected with the rAAV8 vector via PV (8PV). SC injection achieved approximately half of the levels with both AAV9 and AAV8 vectors, but still expressed over 100 |[mu]|g/ml of hFIX in plasma at this dose. Liver transduction in females was lower than males by |[sim]|30% with both serotypes as seen with rAAV2 vectors in this mouse strain. Interestingly, TV and IP injections of the rAAV9 vector showed slower kinetics of hFIX expression compared to the PV injection, even though any of these routes achieved a comparable level of expression 2 weeks-post injection. Such distinct expression kinetics depending on the route of vector administration was not observed with rAAV8 vector. In the second experiment, we injected C57BL/6 mice with rAAV9 vectors expressing a lacZ marker gene; i.e., AAV9-EF1|[alpha]|-nlslacZ or AAV9-CMV-lacZ via TV at two different doses, 3.0|[times]|10e11 and 1.8|[times]|10e12 vg/mouse (n=2 per group, total 8 mice for 4 groups). Development of anti-|[acirc]| galactosidase antibody was monitored by ELISA. The antibody was not detected in any of the 8 mice 7 days post-injection, but detected in 3 of 4 mice injected with AAV9-CMV-lacZ 10 days post-injection, when all the 8 mice were sacrificed for tissue distribution analysis. Liver transduction efficiency with the CMV vector was 3% at 3.0|[times]|10e11 and reached 90% at 1.8|[times]|10e12 vg/mouse. The tissue distribution pattern with rAAV9 vectors was similar to that with rAAV8 vectors, in that, in addition to the liver, they transduced heart, skeletal muscle and pancreatic acinar cells with high efficiency. Of note is that rAAV9 had greater transduction efficiency in the heart than rAAV8, and 3.0|[times]|10e11 vg/mouse was enough to transduce all the cardiomyocytes via TV injection of rAAV9. Thus, rAAV9, as well as rAAV8, is a robust vector for gene therapy applications.

rAAV vectors integrate into host chromosomal DNA infrequently. However, we still may need to consider the possibility of adverse effects caused by rAAV vector integration because 1) rAAV2 vector integrations in animals accompany host chromosomal deletions or translocations, and occur preferentially in or near gene regulatory sequences; and 2) 3 to 4 % integrations occur in or near cancer-related genes. In order to further address this issue and understand the biology of rAAV vectors in vivo, we investigated rAAV8 vector integration in normal and DNA-PKcs-deficient scid mice. Scid mice were chosen because DNA-PKcs has been shown to be involved in rAAV vector genome processing. Here, we developed a novel strategy for isolation of a large number of rAAV proviruses directly from vector-transduced animal tissues with high efficiency and reliability, and without any selective pressure. The salient features of the strategy are: 1) the use of a shuttle rAAV8 vector carrying an ISceI site and AmpR/Ori (AO) cassette, 2) physical separation of integrated and extrachromosomal vector genomes following ISceI digestion of tissue DNA; 3) removal of unwanted genomic DNA by digestion with a DNA conformation-dependent nuclease; 4) mixing the tissue DNA with yeast genomic DNA at 1:1 ratio at the beginning of the procedure, with yeast DNA serving as a tag sequence to monitor for undesired intermolecular recombination that may arise during the procedure. We injected normal and scid mice with 7.2|[times]|10e12 vg of this rAAV8 shuttle vector via the tail vein, and made rAAV8 integration provirus libraries from transduced mouse liver DNA. Transformation of bacteria with |[sim]|0.3 |[mu]|g liver DNA generated over a thousand colonies in each library. Initial sequencing results of 121 and 69 clones from normal and scid mouse libraries demonstrated that 23 (19%) and 52 (75%) of the sequenced clones carried rAAV8 vector-cellular DNA junctions, respectively. Importantly, we have not seen any intermolecular recombination between rAAV8 vector and yeast genomes, establishing the high reliability of this strategy. A preliminary analysis suggested that integration sites in scid mice are more dispersed than in normal mice. In addition, we found that a majority of rAAV8 genomes in scid mouse liver form extrachromosomal double-stranded |[ldquo]|dog bone|[rdquo]| structures, i.e., with both terminal hairpin loops closed. Such dog bone forms are present at undetectable levels in rAAV2-injected mouse livers, but may represent intermediates toward various vector forms. Thus the isolation of a large number of rAAV proviral genomes from quiescent somatic cells in animals is feasible by our novel strategy, and high-throughput rAAV integration site analyses in normal and scid mouse tissues and detailed characterization of the dog bone forms will provide new insights into the mechanisms of rAAV vector transduction and integration in animal tissues.