Background: Inflammation plays a crucial role in the pathogenesis of congestive heart failure (CHF). Enhanced plasma levels of proinflammatory cytokines in patients with CHF have been demonstrated by ...many other groups. TNF-α targeted approach was not sufficient to disrupt the activated network of inflammatory mediators in CHF. IL-10 exerts pleiotropic cardioprotective effects, such as anti-inflammatory effect, and improvement of the endothelial cell function. Previously, we reported that systemic expression of IL-10 ameliorated hypertension as well as cardiac hypertrophy in the Dahl salt-sensitive (DS) rats. In this study, we further investigated LV systolic function and remodeling to examine the preventive effects of IL-10 on CHF in the DS rats.Methods: We constructed AAV type 1-based vectors that express rat IL-10 (AAV1-IL-10) or EGFP (AAV1-EGFP) driven by the CAG promoter. The DS rats were intramuscularly injected with the vector (1 × 10 12 genome copies/body, n=10 each) at 5 weeks old. Systolic blood pressure (sBP) was measured by the tail-cuff method every 2 weeks. Transthoracic echocardiography was performed at 5, 11, and 19 weeks old. At 20 weeks old, heart tissue was examined under the light microscope following H&E, Azan-Mallory staining, and immunohistochemistry for ED-1.Results: Six weeks after single intramuscular injection of the vectors, the serum concentration of IL-10 significantly increased in the IL-10-transduced group compared to that in the EGFP-transduced group (418 vs. 0 pg/ml, p<0.0001). TNF-α concentrations in serum and TGF-β 1 levels in the heart decreased at 20 weeks old in the IL-10-transduced group (0 vs. 7.1 pg/ml, p<0.005; 63.5 vs. 174.3 pg/mg protein, p<0.05). Four weeks after injection of the vectors, sBP of the IL-10-transduced group significantly decreased compared to that of the EGFP-transduced group. This sBP reduction persisted over 2 months (at 18 weeks old; 175 vs. 231 mmHg, p<0.0001). At CHF phase (19 weeks old), LV systolic dysfunction (percent fractional shortening; 59.2 vs. 32.9, p<0.0001) and LV dilatation (LV end-diastolic dimension; 7.3 vs. 8.8 mm, p<0.0001) improved in the IL-10-transduced group. Importantly, histological examination revealed that fibrotic changes and macrophage infiltration significantly increased in the heart of the GFP-transduced group. However, these cardiac remodeling were abolished in the IL-10-transduced group. Furthermore, survival rate also improved at 20 weeks old in the IL-10-transduced group (100 vs. 0%).Conclusion: AAV vector-mediated systemic expression of IL-10 in the DS rats effectively improved inflammation, hypertension, and CHF. Protein supplementation therapy through AAV vector- mediated systemic IL-10 expression would be a promising strategy to prevent the development of cardiac remodeling and CHF in patients with genetic predisposition.
Vectors using AAV8 capsid have shown remarkable results in liver-directed gene transfer in mice. However, the utility of AAV8 vectors in larger animal models have scarcely been described. Here we ...report our results with mice and non-human primates (cynomolgus macaque) to test the usefulness of AAV8 vectors for human applications. As a transgene, we chose macaque coagulation factor IX (FIX) with a mutation at position 262 (macFIXT262A). Based on our previous study, this molecule carries minimal alteration and can be detected with a monoclonal antibody against human FIX, and an assay system utilizing this antibody has been established to quantitate macFIXT262A even in the presence of macaque FIX (J of Thromb and Haemost 2: 275-80, 2003). A stock of AAV8 vector encoding macFIXT262A driven by human alpha1-antitrypsin (hAAT) promoter with a liver specific enhancer was prepared. When the vector was injected into C57BL/6 mice intraportally at 1 × 10 10 vg/body, plasma concentration of the transgene showed more than 100 % of the normal level. When the same vector was injected into a young adult male macaque at a dose of 1 × 1012 vg/kg, plasma concentration of the transgene was detectable throughout the observation period, but not recognizable as therapeutic level (< 0.1%). The efficacy was again tested in another male at a higher titer of 1 × 1013 vg/kg, and resulted in a similar outcome with a slightly higher level. Macaques were extensively immunosuppressed with FK506 and cyclophosphamide until 8 weeks after injection. To better understand these results, potential factors affecting transgene expression were analyzed. Neutralizing antibody against AAV8 capsid was not detectable before injection. Antibody against transgene product was not recognizable. At present, none of the factors inhibiting transgene expression is identified, implying a species-specificity of the efficacy of AAV8 vectors.
The ability to stably introduce genetic material into primate embryonic stem (ES) cells could allow their broader application. We previously derived ES cell lines from cynomolgus monkey blastocysts. ...In this study, we examined lentiviral gene transfer into cynomolgus ES cells. When cynomolgus ES cells were transduced once with a simian immunodeficiency virus (SIV)-based lentivirus vector encoding the green fluorescent protein (GFP) gene, most cells (around 90%) fluoresced, and high levels of GFP expression persisted for 5 months without selection procedures. In addition, high levels of GFP expression were observed during embryoid body formation. On the other hand, transduction of mouse ES cells with the SIV-based vector resulted in lower gene transfer rates, implying that SIV vectors can transduce primate ES cells more efficiently than mouse ES cells. The use of GFP as a reporter gene allows direct and simple detection of successfully transduced ES cells and facilitates monitoring of ES cell proliferation and differentiation both in vitro and potentially in vivo. Furthermore, this highly efficient gene transfer method allows faithful gene delivery to primate ES cells with potential for both research and therapeutic application.
Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to ...current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x1012 gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x1012 gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.
After a considerable dispute, it was shown that cells with hematopoietic activity in muscles are derived from hematopoietic organ. Transdifferentiation is not a frequent event, if any, and such a ...phenomenon cannot be applied to tissue regeneration easily. To achieve a breakthrough, we explored the possibility that genetic manipulation may enhance the efficiency of transdifferentiation. In this regard, there is a notable report that transient overexpression of a homeobox-containing transcriptional repressor Msx1 in muscles generated abundant mononuclear cells (MNCs) capable of differentiating into myotubes, chondrocytes, adipocytes and osteocytes. That is, enforced Msx1 expression caused dedifferentiation of myotubes, and subsequent Msx1 suppression induced redifferentiation (Odelberg SJ et al, Cell 103:1099). Recently, we found that similar dedifferentiation-redifferentiation events also occur in vivo after transient Msx1 expression in muscles using adeno-associated virus (AAV) vectors (Endo T et al, manuscript in preparation). Since virtually all of AAV vector-mediated transgenes exist as episomes, they gradually disappear as the host cells divide. In the present study, we took advantage of this feature of AAV vectors; muscle-derived MNCs would lose Msx1 transgene through cell divisions after dedifferentiation, and a proper differentiation cue might redirect these undifferentiated cells into the hematopoietic lineage as well. AAV vector expressing Msx1 (AAV/msx1) was injected into tibialis anterior muscles of C57BL/6 mice. Flow cytometric analysis revealed that MNCs from AAV/msx1-treated muscles contained a considerable number of cells expressing hematopoietic stem cell markers. CD34−/c-Kit+ cells (1.3±0.9% of MNCs in control muscles) were increased in AAV/msx1-treated muscles (13.6±6.0% at 4 weeks). CD45+/Sca-1+ cells (2.4±0.4% in control muscles) were also increased in the AAV/msx1-treated muscle, peaking at 2 weeks (36.0±8.1%; P =.01). To evaluate hematopoietic activity, MNCs were cultured in methylcellulose medium containing stem cell factor, IL-3, IL-6 and erythropoietin. After AAV/msx1 injection, colony-forming cells in the muscles were gradually increased, reaching a peak at 3 weeks (48.9±24.1/muscle), in contrast to very few progenitors detected in control muscles (1.4±3.0/muscle; P <.01). Finally, in vivo reconstitution activity was evaluated by transplanting MNCs from AAV/msx1-treated muscles of Ly5.2 mice to irradiated congenic mice. About 3x105 muscle-derived cells (Ly5.2) and 2x105 fresh Ly5.1 bone marrow cells were cotransplanted into lethally irradiated Ly5.1 recipients. Among 5 mice transplanted, a robust engraftment of Ly5.2 cells was observed in 2 animals. These transplants showed a very high chimerism of Ly 5.2 (55.2% at 8 months and 41.7% at 6 months, respectively). Furthermore, in the secondary bone marrow transplantation from the former mouse to a Ly5.1/5.2 heterozygous recipient, the donor cell chimerism was even higher (82.9% at 7 months). These results suggest that enforced Msx1 expression can reprogram muscle cells into multipotential cells capable of differentiation into the hematopoietic lineage. This novel technology would be applied to the treatment of acquired bone marrow failure using genetically normal hematopoietic stem cells derived from the patient muscle.
We report on seven chronic myelogenous leukemia (CML) patients who received autologous bone marrow transplantation (ABMT) using bone marrow (BM) cells while at the chronic phase (CP) under the ...various treatments. Of the seven patients, four progressed to accelerated phase (AP) in 83-248 weeks after onset and three patients entered blastic crisis (BC) in 84-171 weeks after onset. All patients received high-dose chemoradiotherapy followed by infusion with 11.3 +/- 12.1 x 10(7) (average +/- S.D.) of bone marrow mononuclear cells (BM-MNCs)/kg IFN-alpha was resumed shortly after platelet recovery. Of the four patients in AP, one developed a recurrence of blastoma in 7 weeks, one progressed to second AP in 138 weeks after ABMT and two patients have survived the second CP for 159 and 330 weeks since ABMT, respectively. One of them achieved the complete disappearance of Ph1-positive metaphases for 33 weeks after ABMT. Of patients who received AMBT in BC, three relapsed within 8 weeks and died in 9, 17 and 58 weeks after ABMT, respectively. Hematological recovery was delayed in four patients. Therefore, we retrospectively re-evaluated the number of BM-MNCs collected through 50 procedures from 40 patients with CML-CP. The total MNCs obtained from 30 collections under IFN-alpha treatment was 27.4 +/- 30.9 x 10(8) cells (average +/- S.D.), being significantly lower than that obtained from 20 collections in pre-treatment state or with single chemotherapy other than IFN-alpha treatment (81.8 +/- 68.2 x 10(8) cells) (P < 0.005). The total number of MNCs correlated to white blood cell (WBC) count at BM collection (P < 0.01), which was also lower in the IFN-alpha(+) group than in the IFN-alpha(-) group (7.2 +/- 5.7 and 25.6 +/- 32.3 x 10(9)/l; P < 0.005). Our findings suggested that ABMT with the use of a sufficient number of progenitor cells might be helpful to CML patients in early AP and reach in extended periods of second CP. In addition, we suggest that BM collection is required before the start of IFN-alpha therapy because the total number of BM-MNCs correlated to the WBC count, which might be lower in IFN-alpha treatment.
AAV8-based vectors are shown to be especially efficient in transducing liver and muscle and currently considered as one of the most promising vector system. In this study, we investigated tissue ...distribution of expression using AAV8-based vectors after intramuscular (IM) injection and other routes of delivery. For this purpose, AAV8-based vectors carrying luciferase gene (Luc) under CAG promoter was administered into tibialis anterior (TA) muscles of C57BL/6 mice. In vivo bioluminescence imaging analysis using IVIS system revealed robust transgene expression not only in lower limbs (around TA) but also in the upper abdominal region (suggesting liver). Analysis by ex vivo bioluminescence imaging after sacrifice confirmed expression in liver as well as TA muscles. To verify whether these findings are unique to AAV8, AAV1- and AAV2-based vectors were tested in the same manner. When AAV1- or AAV2-Luc was injected within TA muscles, Luc expression was confined to the injected sites. The level of transgene expression observed in liver after AAV8-Luc injection was comparable to that observed in lower limbs after AAV1-Luc injection. To substantiate ectopic expression after intramuscular injection, vector genomes within the tissues were quantitated at early time points. Vector genome copies were sharply decreased in muscle and increased in liver at 24 hours after injection in the case of AAV8-based vectors. When AAV8-Luc vector was administered intravenously (IV) or intraperitoneally (IP), luciferase expression was confined to liver. A sex-related difference in luciferase expression in liver was observed regardless of the route of administration. In addition to adults, we compared tissue distribution of expression in neonate by the routes of administration of AAV8-Luc vector. Surprisingly, liver specific Luc expression was observed only after IV injection; in IM and IP administration, transgene expression was confined to lower limbs and peritoneum, respectively. Moreover, sex-related difference in transgene expression was not observed in any tissues in neonatal injection. These data suggest remarkable affinity of AAV8-based vectors to liver, and will be useful in designing strategies for gene therapy using AAV8-based vectors in adult and neonate.
We have shown that intra-adipose tissue injection using AAV1 vectors resulted in tissue-specific transgene expression in db/db mice. To attain a more efficient expression, we tried the same method ...using AAV8-based vector (AAV8-CMV-Epo). Remarkably higher plasma Epo concentration was observed. When a vector encoding human Factor IX (AAV8-CMV-hFIX) was used at a dose of 2 × 10 11 vg/body, more than 10% equivalent of normal human concentration was observed and considered as therapeutic level. In order to confirm tissue specific expression, transduced adipose tissues were surgically removed and plasma Factor IX concentration was pursued one week later. Surprisingly, approximately two-third of plasma concentration remained after total elimination of target tissue. Copy number of hFIX mRNA within the tissue correlated well with the plasma Factor IX concentration, supporting the contribution of both tissues. In vivo imaging analysis by IVIS system suggested transgene expression within liver when AAV8 vector encoding luciferase was used for adipose tissue transduction. Organ specific expression analyzed by ex vivo imaging confirmed robust expression in liver rather than adipose tissue. These data suggest extreme tropism of AAV8-based vectors to liver, and we should be careful about overestimating results when tissues other than liver were targeted.
ABH-antigens and A-and B-glycosyltransferase activity were examined in four patients who received an ABO-incompatible bone marrow transplant from HLA-matched donors. ABO phenotype and ABO genotype ...were analyzed using flow cytometry and polymerase chain reaction with sequence specific reaction, respectively. On day 14 after bone marrow transplantation (BMT), the ABO genotype of erythroid burst-forming units was converted into the ABO genotype of the donors in all of the recipients. Afterward, ABO phenotype of red blood cells (RBC) in all recipients completely changed to that of the donors. Rh, P, Diego, Kidd, Duffy and MNSs phenotypes were also changed into the donor's phenotypes. However, in one patient the Lewis blood type did not change into the donor's phenotype. Similarly, using an elution method, ABH antigens of the recipients were detected in RBC in all of the recipients after BMT. In two recipients, the serum glycosyltransferase activities of the recipients were only detected after BMT. These findings indicate that ABH antigens may be released from non-blood cells and adsorbed by RBC, although the possibility of ABO chimeras or unknown mechanisms is not ruled out. Further studies are needed to determine the nature of ABH antigens eluted from RBC of recipients receiving an ABO-incompatible bone marrow transplant.
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