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Background: Our previous studies demonstrated that systemic expression of interleukin-10 (IL-10) decreased atherosclerosis through its anti-inflammatory property. IL-10 prevents the plaque formation in large arteries, but the protective effect on the arterial damage such as hypertensive arteriosclerosis has not yet been demonstrated. To further elucidate the involvement of the inflammatory regulation in the hypertensive arterial damage, we examined the preventive effect of this anti-inflammatory cytokine using the stroke-prone spontaneously hypertensive rats (SHR-SP) which suffer from arteriosclerosis and stroke spontaneously.

Top of pageAbstract Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus (DM), and is the most common cause of postnatal blindness. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific mitogen plays a critical role in angiogenesis of DR. Spontaneously-diabetic non-obese Torii (SDT) rat was recently established as one of the best-suited models to study DR, because they spontaneously develop DM and shows DR that is characterized with intraretinal microvascular abnormalities (IRMA) and tractional retinal detachment (ref. 1). In this study, we evaluated the therapeutic potential of gene transfer of the soluble VEGF receptor, sFlt-1, in SDT rat using adeno-associated virus vector serotype 5 encoding human sFlt-1 gene (AAV-sFlt-1). Vectors were injected into the subretinal space of 27-week-old SDT rats using 39-guage nyron needle under a surgical microscope. One eye was designated as the “treated side” that received AAV-sFlt-1 in addition to vectors encoding β-galactosidase gene (AAV-LacZ), whereas the other was “control” that received only AAV-LacZ. All of the SDT rats showed hyperglycemia throughout the study, and HbA1c levels were high at the end of the study (9.4 ± 0.95 %: means ± S.D.). Thirty weeks after vector administration, the progression of DR was assessed by fluorescein angiography (FAG) using three parameters; avascular area, extensive hyperfluorescein and arterial narrowing, without the information of injected vectors. Progression of DR in treated eyes was significantly less than that in the control eyes. X-gal staining of the retinal tissue demonstrated the persistent transgene expression for 30 weeks. Moreover, plasma levels of sFlt-1 were undetectable by ELISA, and no adverse effects of sFlt-1 gene therapy were observed throughout the study. These results indicate that local sFlt-1 gene transfer inhibits IRMA of SDT rat, and offers a powerful therapeutic potential to the management of DR.

Currently, skeletal muscle and liver are the preferred target of gene transfer to supply transgene product into systemic circulation. Adipose tissue holds a number of attractive features as a target of gene transfer; it is one of the most abundant tissue in the body, and are designed to secrete various proteins physiologically, making it suitable to supply transgene product into systemic circulation. Of note, the transduced tissue can be safely removed without any sequelae in case of unexpected events. However, efficient transduction of adipose tissue has not been feasible by conventional methods. In order to develop a practical method, we tested the enhancement effect of the excipients upon gene transfer. At first, AAV vectors encoding LacZ were administered into adipose tissue of Db/Db mice. Widespread β-Gal expression was observed when a bio-compatible surfactant was included in the vector solution. To validate the efficacy of this method, vectors encoding mouse erythropoietin (Epo) were utilized. Two weeks following vector injection, significant concentrations of plasma Epo were observed and the levels were almost comparable to that of muscle- or liver- mediated gene transfer. Plasma Epo concentrations kept similar levels during the observation period thereafter. Increased blood hemoglobin levels warrant biological activity of adipocyte-derived Epo. Efficient expression was also confirmed by both immunofluorescence staining of the adipose tissue using anti-Epo antibody and RT-PCR. In addition, following complete removal of transduced adipose tissue, increased plasma Epo concentrations returned normal, suggesting that other tissues were not transduced by this method. Although the precise mechanisms of this phenomenon are yet to be analyzed, our method leads to efficient gene transfer into adipocytes, adding a novel choice for the supplemental gene therapy approaches.

To overcome the low efficiency of gene transfer into hematopoietic stem cells, we developed a novel system for selective expansion of transduced cells. The system utilizes “SAGs (selective amplifier genes)” which encode fusion proteins consisting of a growth-signal generator and its molecular switch. First, a fusion gene encoding a chimeric receptor between G-CSF receptor (GCR) and hormone-binding domain of estrogen receptor (ER) was constructed. We deleted the G-CSF-binding domain from the chimeric gene and introduced a mutation (Y703F) to block the differentiation signal. In addition, a mutant ER (TmR), which binds to 4-hydroxytamoxifen (Tm), was also used in place of a wild type ER. When the murine bone marrow cells transduced with ΔY703F-GCRTmR gene were transplanted into lethally irradiated mice, the proportion of transgene-positive leukocytes in peripheral blood increased in response to Tm administration. In a cynomolgus macaque model, half of six monkeys that received the SAG showed in vivo increase of transduced hematopoietic cells following E2 or tamoxifen administration. Recently, we are developing the second-generation SAGs, which encode chimeric receptors between extracellular and transmembrane portion of erythropoietin receptor (EPOR) and intracellular portion of GCR or thrombopoietin receptor (Mpl). We transduced human cord blood CD34+ cells with EPO-driven SAG (EPOR-GCR or EPOR-Mpl). The cells transduced with EPOR-Mpl proliferated most efficiently. The EPOR-Mpl also expanded and preserved c-Kit+ cells most efficiently. Preliminary experiments suggest that the EPOR-Mpl conferred potent EPO-dependent growth advantage on the transduced hematopoietic cells in vivo in a murine system. The SAG system may circumvent the low efficiency of gene transfer.

One of the therapeutic strategies for relapsed leukemia after allo-BMT is donor lymphocyte infusion (DLI), in which graft-versus-leukemia (GVL) reaction via allo-reactive cytotoxic T lymphocytes (CTL) is a major factor contributing to the eradication of malignant cells. However, the most serious obstacle is life-threatening GVHD. One strategy to regulate harmful CTL activity is to incorporate a suicide gene into donor lymphocytes prior to DLI, allowing on-demand removal of these cells when severe GVHD occurs. For this purpose, HSV-TK gene has been used in combination with ganciclovir, but there are several problems in this system. We have developed a distinct apoptosis induction system by using a gene encoding Fas-estrogen receptor chimeric molecule (MfasER gene from Dr. A. Kakizuka) for eliminating transduced cells. We investigated the feasibility of using MfasER gene to control CTL activity. A murine CTL line, CTLL-2, was used as an effector in model experiments. MfasER gene transfer itself did not affect the viability and cytotoxic activity of CTLL-2. The addition of estrogen into the culture media eradicated more than 90% of the transduced CTLL-2 with significant decrease in the cytotoxicity. Considering in vivo applications, the MfasER/estrogen system has several desirable characteristics such as least immunogenicity of the fusion protein, low toxicity of the reagent, and cell cycle-independent apoptosis induction. Furthermore, Fas-mediated apoptosis pathway would work more effectively in activated T-lymphocytes than in resting cells, which provides a rationale for this approach.

Glycine decarboxylase, a constituent of the glycine cleavage system, in patients with either nonketotic or ketotic hyperglycinemia (NKH and KH) was examined using an anti-chicken glycine decarboxylase antibody. Patients with NKH who have lesion in glycine decarboxylase are differentiated by its expressed level in the liver. One group is cases of the neonatal onset type who have neither activity of the enzyme nor protein reactive to the antibody. The other is a case of the late onset type who shows low but detectable activity of the enzyme and the desirable amount of the immunoreactive material. In the liver of a patient with KH not showing the appreciable activity of H-protein, ubiquitous amount of protein reactive to anti-H-protein IgG is detected and amount of glycine decarboxylase has also been lowered. It is suggested that several mechanisms may be involved in determining the expressed level of glycine decarboxylase in patients with hyperglycinemias.

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.