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Summary. Background: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Methods and Results: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. Conclusions: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

Objective The homeobox B4 ( HoxB4 ) gene promotes expansion of hematopoietic stem cells (HSCs). However, frequent development of leukemia in large animals due to retrovirally transduced HoxB4 gene has been reported. To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/ΔP), which enabled clearance of the vector and transgene shortly after transduction. We tested the SeV/ΔP vector expressing the HoxB4 gene (SeV/ΔP/ HoxB4 ) for the ex vivo expansion of human cord blood CD34 + cells (HSCs) using a sheep in utero transplantation assay. Materials and Methods Human HSCs were ex vivo−expanded by transduction with SeV/ΔP/ HoxB4 vector and transplanted into the abdominal cavity of fetal sheep. The engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony-forming unit assays. Results After transplantation, the HoxB4 -transduced HSCs contributed to longer-period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group ( p HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4. The SeV/ΔP/ HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post-transplantation. Conclusions The SeV/ΔP vector would be suitable for transient expression of HoxB4 in human CD34 + cells. In addition, the SeV/ΔP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors.

Chagas' disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8(+) T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8(+) T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-gamma (IFN-gamma) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-gamma production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas' disease.

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.

We have previously shown that the lung can be used as “factory” for production of secreted proteins, which are released into the circulation, using Sendai virus-mediated gene transfer. However, gene expression was transient and repeated administration of the vector was not feasible due to induction of immune responses. We have developed a lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN) to allow efficient transduction of the airways. The unique features of rSIV.F/HN, namely stable expression for >20 months after a single dose and efficient transduction after repeated administration, make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV.F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n=5-6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p

Nonstoichiometry of the perovskite-type solid solutions La0.9Ca0.1Cr1−yAlyO3−δ was studied by high-temperature gravimetry under controlled P(O2) atmospheres of 1–10− 23 bar at 1073–1273 K. The observed data were described by a regular solution-like model for the randomly distributed defects of VO¨ , CrCr. , CaLa,, and AlCrX. With the increase in y, VO¨ formation becomes much easier. For y > 0.8, some fraction of CaLa, becomes surrounded only by AlCrX and VO¨ remains around such CaLa, up to high P(O2) to reduce the maximum oxygen content below 3.000.

The nuclear membrane is a tight barrier against the delivery of therapeutic genes into non-dividing tissue cells. Overcoming this barrier with the aid of peptidic nuclear localization signals (NLS) is crucial for improving the performance of synthetic gene-delivery vehicles. In this article, we examine the nuclear transport of lambda phage particles displaying various peptides containing the minimum NLS of SV40 T antigen on their surface. As the minimum NLS (PKKKRKV) is a binding domain to importin alpha, recombinant proteins and molecular conjugates containing this peptide accumulate into the nucleus efficiently. However, we find that the C-terminal and N-terminal structures besides the minimum NLS profoundly affect the efficiency of the nuclear transport of the phage particles as well as their binding capacity to importin alpha: either truncation of a few amino acid residues from the C-terminus or the replacement of the N-terminus with a FLAG- or c-myc-tag abolish both of these biological activities. The structure of the optimized NLS is unpredictable from conventional protein transport assay and from the structural analysis in silico. Our results reveal that the objects with 50 nm in diameter can pass through the nuclear pore complex when the optimized NLS is displayed at a sufficient density on their surface.

The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2–30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (

We previously developed “selective amplifier genes (SAGs)” which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34+ cells than the GCR-type SAG. One mutant Mpl-type SAG (ΔGCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.

The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.

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.

We have previously shown that recombinant Sendai virus (SeV) produces efficient in vivo airway epithelial gene transfer. The ability to produce therapeutic levels of circulating proteins following noninvasive gene transfer would have widespread clinical application. Here, we compared nose, lung, and skeletal muscle for the ability to produce circulating levels of the secreted mouse antiinflammatory cytokine interleukin-10 (IL10) following SeV-mediated gene transfer. High levels of serum IL10 were obtained from each site with a potency order of lung > nose > muscle for a given viral titer. Serum levels from each site were within the likely required range for anti-inflammatory effects. The combination of a high-efficiency gene transfer agent (SeV) and sites that can be assessed noninvasively (nose or lung) may circumvent several current challenges to gene therapy.

The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant lambda phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tat-phage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.

We are developing lung gene transfer vectors to direct abundant, sustained transgene expression in three therapeutic compartments: (i) within lung cells to treat lung disorders such as cystic fibrosis (CF), (ii) secreted from lung cells into the lung lumen to treat viral infections and airway diseases such as alpha1 anti-trypsin deficiency, (iii) secreted from nasal and lung cells into the circulatory system to treat hematologic and a range of other disorders. Such diseases are associated with a high burden of care often involving frequent antibiotic treatments, physiotherapy and supportive systemic infusions; and considerable patient morbidity and mortality. We have developed an efficient lung gene transfer platform based on a European and US Pharmacopoeial compliant, minimal recombinant SIV lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN). Initially, production of rSIV.F/HN vectors was established in a serum-dependent, calcium phosphate-mediated, transient transfection process. Here we report the development of a scalable, animal-free, cGMP-compliant suspension cell culture based upstream production (USP) method. Serum-free media screening identified a suspension cell growth conditions that support efficient (>70%) multiple plasmid transient transfection of HEK293T cells with a range of cGMP-compliant liposomal and polymeric gene transfer agents. Small volume (40–400mL) studies in Erlenmeyer shake flasks and Design Of Experiments statistical approaches identified robust growth and transfection conditions that yield ≥5e6 TU/mL of unpurified rSIV. F/HN vectors. These parameters supported transition to 1L and 5L scale WAVE bioreactor cultures, where virus yield is maintained and plasmid DNA usage (a high proportion of the cGMP cost of goods) has been reduced to 0.33mg/L. F/HN pseudotype specific DNA removal and virus activation steps are incorporated into the downstream purification (DSP) method which relies on anion-exchange membrane virus capture and hollow-fibre UF/DF for vector purification and final formulation. In 56 independent, pH-controlled WAVE bioreactor cultures, purified yield exceeds 2.0e9 TU/L for a range of reporter gene constructs with a variety of promoters and 1.2e9 TU/L for a range of CFTR constructs (p>0.05; n=42/14 respectively). The final product directs abundant in vivo airway gene transfer, transducing 14.1% epithelial cells in the murine lung (p

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) using this lead candidate vector. Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.

This report describes the isolation of a cDNA encoding a novel human Gal beta (1-3/1-4)GlcNac alpha 2,3-sialyl-transferase involved in the biosynthesis of the sialyl Lewis x determinant (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A cDNA library of the human melanoma cell line WM266-4 was constructed in an Epstein-Barr virus-based cloning vector. Selection of the B-cell line Namalwa expressing transfected cDNAs in the presence of the cytotoxic lectin Ricinus communis agglutinin 120 gave a cDNA encoding a protein with type II transmembrane topology, as found for mammalian glycosyltransferases. The use of this lectin, which is specific to galactose residues (especially the Gal beta 1-4GlcNAc structure), originates from our prediction that the modification of the Gal beta 1-4GlcNAc structure (a backbone of the sialyl Lewis x structure) by glycosyltransferases may increase the levels of resistance to this lectin. Comparison of this cDNA sequence with those of three other cloned sialyltransferases revealed two conserved regions shared by all four enzymes. Expression of the COOH-terminal catalytic domain of this protein showed alpha 2,3-sialyltransferase activity with substrate specificity different from that of CMP-N-acetylneuraminate:N-acetyllactosaminide alpha-2,3-sialyltransferase (Gal-beta 1-3(4)GlcNAc alpha 2,3-sialyltransferase, EC 2.4.99.6). Furthermore, expression of this cDNA in Namalwa cells increased the level of sialyl Lewis x antigens. The cloning approach based on lectin resistance may be useful for the isolation of cDNAs encoding other mammalian glycosyltransferases.

A protease/anti-protease imbalance is a characteristic feature of inflammatory lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). However, alpha-1-antitrypsin (AAT) enzyme replacement therapy (ERT) trials have not shown conclusive evidence of a therapeutic benefit. This may be due to inefficient protein delivery to the lung and/or the half-life of the protein. The UK Cystic Fibrosis Gene Therapy Consortium (GTC) has pseudotyped a simian immunodeficiency viral vector with the Sendai virus F and HN proteins (rSIV.F/HN) and selected an internal synthetic promoter/enhancer consisting of the elongation factor 1α promoter and the CMV enhancer (hCEF) for efficient transduction of airway epithelial cells. Here, we assessed whether transduction of murine lungs with rSIV.F/HN with a human AAT transgene generates therapeutic levels of AAT. Mice were transduced with rSIV.F/HN-hCEF-AAT (2e8 TU/mouse) by nasal instillation and culled 10 days after transduction. AAT levels in lung homogenate and broncho-alveolar lavage fluid (BALF) were at least 3 logs above controls (p

Top of pageAbstract Recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane and leads to high, but transient gene expression. The treatment of chronic diseases may require repeated administration of the virus. We have previously shown that three repeat administrations of recombinant Sendai virus (DF/SeV) did not lead to measurable luciferase (lux) expression after the third administration. However, as part of these experiments we noted that even after one administration, lux expression was lower than expected for a given virus dose. Sequence analysis revealed that the lux gene contained sequences that may be recognised by SeV as |[ldquo]|stuttering|[rdquo]| sequences and may interfere with gene expression. In an attempt to improve SeV-mediated lux expression we introduced several silent mutations to modify these putative stuttering sequences. SeV-mediated lux expression in the lung was increased 2.5-fold (n=10, p

[Background] Antigen-presenting cells, particularly dendritic cells (DCs) are important elements of antigen-specific adaptive immunity. Antigen-loaded DCs have now been used as a vaccine to improveimmunity against various cancer. However, the efficacy of therapeutic vaccination against cancer using DCs in the current clinical studies has been limited because of inadequate DC treatment, lack of established administration routes of DCs or uncomfortable environment to DC around tumor. We here report a novel method to activate DCs ex vivo by use of recombinant Sendai virus (SeV) for cancer immunotherapy. In vitro treatment with SeV led DCs to highly activated levels as indicated by surface markers, such as co-stimulatory molecules. We examined efficacy of SeV-activated DCs against murine squamous carcinoma (SCC VII).

Although intracerebral grafting has become established as a new strategy for the treatment of Parkinson's disease, there are many problems regarding such grafts. We focused on the grafting of primary skin fibroblasts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing cDNA of human tyrosine hydroxylase (TH) type 1 (LTHSNL) or of cytomegalovirus promoter (CTHSNL) as a foreign promoter. In these genetically modified fibroblasts, L-DOPA production and release were analyzed in vitro by immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Being supplemented with the biopterin (BH4:(6R)-L-erythro-tetrahydrobiopterin) cofactors required for TH activity, these cells produced and released L-DOPA into the culture medium. When CTHSNL and BH4 were combined, L-DOPA production increased with time, regardless of the number of cell passages, or the duration of liquid nitrogen freezing. This suggests that the foreign gene (THcDNA) containing retrovirus vector integrates into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with a retrovirus vector system containing the TH cDNA may be promising grafts for transplantation and gene therapy in Parkinson's disease.

Since the prognosis of squamous cell carcinoma (SCC) in the head, neck, and esophagus has shown only a little progress in the last decade, the development of novel therapeutic strategies has been much desired to treat patients with advanced SCC. As a candidate, dendritic cell (DC)-based immunotherapy has been tested, but still requires modification to improve clinical outcomes. We here show that non-transmissible Sendai virus (SeV/dF)-activated DCs (DCs/SeV/dF) has strong antitumor effects against an orthotopic model of murine SCC, namely a less immunogenic SCCVII tumor. SeV/dF showed high transfection efficiency at low MOI to murine bone marrow-derived DCs (mBM-DCs). Higher viral doses of SeV/ dF-GFP did not result in a significant cytopathic effect. SeV/dF leads mBM-DCs to upregulate costimulatory molecules such as CD40, CD80, CD86, and MHC class II. We subsequently assessed the effects of SeV/dF-null transfection on endo-/phagocytotic activity of mBM-DCs. Their activity in taking up FITC-dextran was assessed at various time points after stimulation. Unlinke in DC/ LPS, endo-/phagocytic activity of DCs was not impaired by SeV/ dF. Intratumoral injection of DCs/SeV/dF resulted in a marked and representative inhibition of the tumor, even when the tumors were well-vascularized. The antitumor effects of intratumor injection of DCs/SeV/dF were not impaired without ex vivo exposure of DCs to tumor lysate, suggesting the potential uptake of tumor antigen in vivo. Our results provide the first demonstration that non- transmissible SeV/dF could be a promising candidate for DC- activator; DC/SeV/dF-based cancer immunotherapy may therefore warrant further investigation with regard to its enhancement of antitumor effects in a clinical setting.

Top of pageAbstract The cytoplasmic RNA vector would be promising for use in gene vaccines to large population of patients or infected persons in urgent or in hospitals of various conditions because of its important genotoxicity-free nature. The candidate intranasal gene vaccines have been developed using Sendai virus (SeV) vector for both infectious and neurodegenerative diseases such as AIDS and Alzheimer's disease (AD), respectively. SeV belonging to the Genus Respirovirus, infects and multiplies its genome copy in most mammalian cells. Its replication is strictly in cytoplasm and independent of nuclear functions of host cells. Moreover, SeV does not have a DNA phase during its life cycle, so SeV-based vectors that express high-level of transgene do not need to be concerned about the transformation of cells by integration of vector materials into the host chromosomes. These properties of the vector enable us to propose the new concepts, CYTOPLASMIC GENE THERAPY and CYTOPLASMIC VACCINATION with RNP-based treatment. For the treatment of AD, the F gene-deleted non-transmissible SeV (SeV/|[Delta]|F) vector carrying amyloid-|[beta]| (A|[beta]|) gene was used. According to the |[ldquo]|Amyloid Cascade Theory|[rdquo]|, the formation of senile plaques through aggregation and deposition of A|[beta]| peptides in the brain is considered as a major cause of the disease. Thus, immune-mediated strategy known as A|[beta]| vaccination has been thought to be a promising therapeutic approach for the treatment of Alzheimer's disease. Single intranasal administration of SeV vector carrying A|[beta]| gene (A|[beta]| 1|[ndash]|43) to the 24 to 25-month-old APP transgenic mice induced the expression of the A|[beta]| peptide in the epithelial cells of the nasal mucosa. Serum antibody level was elevated and the amyloid burdens in the brain were decreased to 10|[ndash]|20% of the control mice after eight weeks treatment. Importantly, no lymphocytes infiltration that into the central nervous system was detected. For the AIDS vaccine, the Gag-expressing SeV/|[Delta]|F vector was used. Macaques vaccinated with DNA-prime/Gag-expressing SeV/|[Delta]|F-boost were challenged intravenously with the pathogenic simian immunodeficiency virus (SIVmac239) that induces chronic disease progression. The SeV boost induced Gag-specific CD4+ T cells, and its level in peripheral T lymphocytes was maintained for more than 40 weeks. The vaccine-induced cytotoxic T lymphocytes (CTLs) controlled SIVmac239 replication well. Thus, the mucosal immunotherapy with SeV vector has the great potential of prevention and treatment for both infectious and neurodegenerative diseases and might be a novel, attractive approach in gene vaccines of coming age.

Extensive monocytes extravasation is seen in infection sites, inflamed tissue and tumor tissues and exhibit a tissue-specific range of functions including phagocytosis, antigen presentation to T cells, and the release of a wide array of cytokines, chemokines, enzymes and nitrogen species. As monocytes have natural tropism to such tissues in each disease condition, these cells can be used as novel gene delivery system to cure the condition. On the other hand, as monocytes are quiescent cells, available gene transfer vectors are limited. Sendai virus (SeV) belonging to the Genus Respirovirus infects and multiplies its genome copy in most mammalian cells, and it has recently been used for the gene therapy vectors, SeV vectors (SeVV), for somatic gene therapy. This vector that expresses high-level of transgene does not have a DNA phase during its life cycle, so it does not need to be concerned about the transformation of cells by integration of vector materials into the host chromosomes, the genotoxicity-free nature. Thus, we used SeVV for gene transfer into monocytes. We first of all studied the gene transduction efficiency of SeVV into human peripheral blood CD 14 positive monocytes using GFP gene as a marker. About 82% of monocytes became GFP positive, whereas only 23% of lymphocytes and 1.8% of granulocytes became positive. The transduction efficiency was peaked at M.O.I.=1. The expression level was at least preserved for 16 hours in vitro. To do the in vivo preclinical experiments, we also transduced cDNA of GFP or GDNF (glial cell line-derived neurotrophic factor) into the peripheral monocytes of Macaca fascicularis monkey and high level expression of these genes were obtained. In vitro monocyte function after gene transduction are now under investigation. SeVV is considered to be promising gene transfer vector for primate monocytes.

We have previously shown that recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane. This is in part due to cholesterol and sialic acid in the apical membrane which are respectively utilized by the SeV fusion (F) and hemagluttinin-neuraminidase (HN) proteins for receptor- mediated cell attachment, as well as the capacity of SeV to penetrate mucus efficiently. In contrast to SeV, which leads to transient gene expression, integrating lentiviral vectors have the theoretical advantage of persistent expression for the life-time of the cell, and ifstem cell integration should occur, for the lifetime of the animal. We have recently succeeded in generating a F/HN-pseudotyped Simian immunodeficiency virus (SiVagm) vector at high enough titre for in vivo application (>109 transduction units (TU)/ml). A F/HN-SiV- GFP vector (4x108 TU/mouse in 100 ml) was slowly perfused (over 75 min) onto the nasal epithelium of C57Bl/6 mice and transduced airway epithelial cells efficiently, via the apical membrane without the need for chemical pre-conditioning. We choose transduction of the nasal epithelium as proof-of-principle because we aim to extend these studies into CF knockout mice (only the nasal epithelium shows the CF defect in this model). We previously reported detection of GFP expression at 1, 20, 30, 90 days (n=3 mice/group) and 220 days (n=1) after transduction. Here, we further extended these studies to both include higher n numbers and, to identify transduced cells. Importantly, 180 days after transduction 8 out of 10 (6 histological sections 1 mm apart/mouse) mice still expressed GFP. Preliminary experiments on a single animal also showed persistent expression 360 days after transduction, and further animals transduced 270 and 360 days ago, are currently being analysed (n=12|[ndash]|15/group). This duration of gene expression is far longer than the predicted life-span of airway epithelial cells of approximately 100 days. One explanation is that F/HN-SiV vector has transduced progenitor or stem cells in the mouse nose. Morphological analysis showed that GFP positive cells were ciliated airway epithelial cells, and cells being in the olfactory epithelium which based on morphological criteria, appeared to be sensory neuronal cells.

Top of pageAbstract Sendai virus (SeV) is currently the only viral vector that transfects airway epithelial cells efficiently in vivo. However, as for any other viral vector repeated administration, leads to a significant drop in gene expression. Induction of immunological tolerance may permit repeated administration of the SeV vector. To achieve this we investigated the induction of immunological tolerance to the CD4 and CD8 T cell immunodominant epitopes of SeV. These peptides (100 mg/mouse) or a control peptide were administered to the lungs of C57Bl/6 mice (female, 6–8 weeks) via “sniffing” 10 days before the first virus administration (dF/SeV-GFP 106 pfu/mouse). 21 days after the first virus administration, mice were repeatedly transfected with dF/SeV. To avoid interference from anti-GFP antibodies, the second transfection was carried out with dF/SeV-lacZ (107 pfu/mouse). Two days after dF/SeV-lacZ administration all animals were culled. The lungs were harvested for βgal quantification and T-cell proliferation in response to the immunodominant epitopes was assessed to determine immunological tolerance. In addition serum was collected to measure anti-SeV neutralising antibodies. Administration of peptide 1 or 2 and 1+2 reduced CD4 and CD8 T-cell proliferation ex vivo after re-challenge with the respective protein by >60%. βgal expression following a second administration of SeV in mice receiving no peptide or the control peptide was 40–50% lower compared to mice that only had a single administration. Administration of the peptides did not elevate expression levels over levels measured in these control groups. This result indicates either that T cell responses need to be further reduced or that immune mechanisms other than T cells play a major role in eliminating SeV.

Sendai virus (SeV) vector is a new class of vector bearing a new concept of “cytoplasmic RNA vector”. This vector based on SeV belonging to the genus Respirovirus of the family Paramyxoviridae, infects and replicates in most mammalian cells, and directs high-level transgene expression. Its replication is independent of nuclear functions and does not have a DNA phase during its life cycle so that the transformation of cells by integration of vector materials into the cellular genome is not a concern. These properties make SeV vectors very promising for application to gene therapy and vaccination via the expression of therapeutic genes and antigens. We have previously succeeded in the recovery of fusion (F) gene-deleted SeV vector (SeV/ΔF), matrix (M) gene-deleted SeV vector (SeV/ΔM), both M and F genes-deleted SeV vector (SeV/ΔMΔF) and other types of the vectors in high titers. F gene-deletion made SeV vector non-transmissible, and M gene-deletion worked well on the vector to become incapable of formation of the particles from infected cells. All of the above SeV vectors maintain efficient infectivity in vitro and in vivo as wild type SeV does. The hemagglutinin-neuraminidase (HN) protein, which mediates the attachment of SeV, is known to be one of the major targets for host immune responsive machineries on SeV infection such as natural killer cell (NK), cytotoxic T lymphocyte (CTL) and neutralizing antibodies. The HN gene-deletion from the genome would be one of the important ways to reduce the immune response against SeV vector. Therefore, the HN gene was further deleted from the genome of SeV/ΔMΔF, and thus SeV/ΔMΔFΔHN was generated. The SeV/ΔMΔFΔHN possesses only nucleoprotein (NP), phosphoprotein (P) and large protein (L)-genes on its genome. All of them are essential for efficient expression of SeV vector by consisting ribonucleoprotein (RNP) complex. The new vector was successfully recovered and propagated in a new packaging cell line expressing M, F and HN proteins by using a Cre/loxP inducible system. The titer of SeV/ΔMΔFΔHN carrying the enhanced green fluorescent protein (GFP) was and above 1×107 cell infectious units (CIU)/ml. This vector showed efficient infectivity and gene expression in various types of cell lines and primary cells in vitro. When this vector was administered into the auricle of the ear of mice (5×106 CIU), a visible transduction was confirmed as in the cases of wild type SeV vector in vivo. The important investigation for immune reaction of this vector is in progress. The SeV/ΔMΔFΔHN vector is the most advanced type of cytoplasmic RNA virus vector and the like Sendai virus vector.

Top of pageAbstract Background and Aim: We previously demonstrated the dramatically high efficiency of recombinant Sendai virus (SeV) for pulmonary gene transfer, 1,000-100,000 fold over that via adenoviruses, and suggested that its possible application for gene therapy of cystic fibrosis (CF) (Yonemitsu Y et al. Nature Biotechnol 2000). As widely known, however, experimental inoculation of SeV exhibits inflammatory and immune responses in rodent lung, frequently resulted in the death of animals. For future directions of pulmonary gene therapy for monogenic diseases, including CF, neonatal gene transfer is ideal, because the lung disease may be in reversible. We recently developed a new and advanced design of SeVs for clinical application, namely F-deficient SeV (SeV/dF), temperature-sensitive SeV/dF (tsSeV/dF), and SeV deleted with all membranous genes (SeV/dFdMdHN). Here we characterized these new constructs for pulmonary gene transfer in neonatal mice, especially referring to toxicity. Method and Results: Recombinant SeVs expressing GFP (2.5 |[times]| 10|[and]|6 pfu in 10|[mu]|l PBS /neonate or 2.5 |[times]| 10|[and]|7 pfu in 100|[mu]|l PBS /adult) was placed into the nasal cavity and the solution was sniffed into the lungs of neonate (day 0) or adult (8 weeks old, female) of ICR (CD-1) mice. The efficiency of gene transfer and the level of gene expression were also monitored using same amount of SeVs expressing luciferase. The peak of gene expression in lung of neonatal mice was at 48 h and decreased gradually, and then almost disappeared at 14 days after infection in all constructs, similar to the finding found in adult mice. Increase of the body weight of neonates transfected with tsSeV/dF or SeV/dFdMdHN, however, was significantly larger than those of animals transfected with wild-SeV or SeV/dF, and the effect was dramatic in use of SeV/dFdMdHN. Mortality rate was dramatically diminished in the mice translated SeV/dFdMdHN (survival rate=100%, n=43) compared with wild-SeV (25%, n=36) or tsSeV/dF (75%, n=44). Assessments of immunological parameters are now underway. Discussion and Conclusion: Our current study revealed that deletion of all membranous genes of recombinant SeV dramatically reduced the toxicity for pulmonary gene transfer in neonatal mice. In conclusion, the potential of pulmonary gene thansfer using SeV/dFdMdHN warrants for further intervention to develop more efficient therapy of neonatal lung diseases including cystic fibrosis.

Recombinant Sendai virus (SeV) is currently the most efficient vector for airway gene transfer. We have previously shown that after a second administration of transmission incompetent F protein-deficient DF/SeV to the lung gene expression was reduced by 60% when compared to a single administration, but was still significantly higher than in untreated mice. Here, we assessed, if this residual level is maintained after further virus administrations. Mice received three administrations one week apart of DF/SeVMtsHNtsPLmut, an F-deficient virus with mutations in the M, HN, P and L protein, that reduce cytotoxicity in vitro (week 1: SeV-GFP, week 2: SeV-GFP, week 3: SeV-lux). Because the immune response, might be depend on virus titre, individual cohorts of animals were treated with either 106, 107 or 108 pfu/mouse (n=20/dose). Luciferase (lux) expression was measured two days after SeV-lux administration. Importantly, repeat administration of DF/SeVMtsHNtsPLmut was well tolerated even at the highest titre. However, Lux expression after three administrations of SeV was significantly reduced compared to single administration, regardless of the virus titre used. A similar study in sheep has been instigated and will serve to indicate whether expression is similarly attenuated in a host in which wild type SeV infection is of no apparent significance. As a prelude to repeat administration the duration of expression after single administration was eastablished. We, therefore, aerosolised sheep with DF/SeV-lacZ (1010 pfu/sheep, n=3) using a Trudell AeroProbe catheter and compared bgal expression in bronchial biopsies (Bx, n=4/sheep from different lobes) and bronchial brushings (Br, n=4/sheep from different lobes) pre-and two days post-transfection. Despite the comparatively small amount of tissue sample bgal expression was significantly (p

Cystic Fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian, and the cystic fibrosis transmembrane conductance regulator (CFTR) gene was identified to its causative gene. Mutations in the CFTR lead to imbalanced ion and water movement across the airway epithelium, resulting in thickened mucus, chronic bacterial infection and inflammation. Such severe pathological changes in airway are responsible for the morbidity and mortality in CF patients. Then, the airway epithelial cells are the important targets for CF gene therapy. However, few vectors can transduce them because of the existence of thickened mucus layer on the apical surface of epithelial cells and/or lack of virus receptors on apical site of the epithelial cells. We have shown that recombinant Sendai virus (SeV), murine parainfluenza virus, can easily penetrate the mucus layer and efficiently transduce the airway epithelial cells from the apical side. In order to improve the lentivirus vector entry, we developed a novel pseudotyped lentivirus vector based on a simian immunodeficiency virus (SIV: SIVagm TYO1) with SeV envelop proteins, fusion protein (F) and hemagglutinin-neuraminidase (HN). Although the SIV vector could not be pseudotyped with the intact F and HN, it could be successfully pseudotyped after the modification of cytoplasmic regions of both F and HN. Thus prepared F/HN pseudotyped SIV vector carrying EGFP transgene was produced by transient transfection in 293T cells to the titers up to 108 transduction units (TU)/ml, and easily concentrated to more than 1010 TU/ml by ultra-centrifugation with the same procedures for VSV-G pseudotyped vectors. Although the VSV-G pseudotyped lentivirus vectors have been used for administration to airways with pre-conditioning treatment using detergents, such treatment should be omitted considering the future clinical trials to human. From this point of view, F/HN pseudotyped SIV vector was evaluated in vivo in the absence of pre-conditioning. In this study, we administrated 100 |[mu]|l vector (4|[times]|108 TU/head) into mouse nasal cavity with syringe driver and observed EGFP expression. Although no or minimum expression was detected by administration of the VSV-G pseudotyped SIV vector, clear, frequent and stable GFP expression was observed in the epithelial cells of nasal cavity in the case of F/HN pseudotyped SIV vector even one month after the transduction. Detailed quantitative results will be summarized in the very near future. Our novel pseudotyped SIV vector with SeV envelope proteins, F and HN, would be of great value as a tool for gene therapy of respiratory diseases such as CF.

Top of pageAbstract Purpose: Recently, we have demonstrated the efficient and stable retinal gene transfer mediated by the non-pathogenic simian immunodeficiency virus (SIV)-based lentivirus vector as well as the therapeutic outcome in some animal models of retinal degeneration using recombinant SIV vectors carrying neurotrophic factors, such as pigment epithelium-derived factor (PEDF) and fibroblast growth factor-2 (FGF-2). Here, we report the acute ophthalmic toxicity following intraocular administration of SIV-PEDF in Macaca fascicularis, as a preclinical safety study. Methods: Eleven Macaca fascicularis (body weight=2.35-5.63 kg) were enrolled in this study. Approximately 20 |[mu]|l of SIV-PEDF (low titer: 2.5|[times]|107 transducing units [TU]/ml, equal to clinically available titer, high titer: 2.5|[times]|108 TU)/ml, and max titer: 1.0|[times]|109 TU/ml, n=3, respectively) or control (balanced salt solution: BSS, n=2) were injected into subretinal space via a glass capillary tube. We undertook an ophthalmic examination including slitlamp biomicroscopy, intraocular pressure (IOP) measurement, fundoscopic examination, and fluorescein anigiography, and assessed transgene expression, retinal function with electroretinogram (ERG) and histology for 3 months. Results: SIV vector was efficiently transferred mainly to the retinal pigment epithelium and transgene expression was detected within 3 months. Transient inflammatory cell infiltration to anterior ocular segment and elevation of IOP were observed in some animals, but not dose-dependent. ERGs including multi-focal ERGs revealed no remarkable change of the retinal functions. Retinas treated with both SIV-PEDF and BSS showed no significant inflammatory infiltration and retinal structural destruction. Furthermore, neither dead animal nor serious side effect was found during experimental course. Conclusions: The current study indicated the acute local and systemic safety of intraocular administration of SIV-PEDF, even if it was given at 40-times higher titer than clinical available titer. We are now conducting the long-termed safety study in non-human primates.

A new class of CYTOPLASMIC RNA VECTORS is thought to be advanced transgene expression systems that overcome recent problem of genetic disturbance by existing vectors. Our Sendai virus (SeV) vector based on SeV belonging to the family Paramyxoviridae, infects and multiplies its copy in most mammalian cells, and directs high-level transgene expression. Its replication is independent of nuclear functions and does not have a DNA phase during its life cycle so that the transformation of cells by integration of vector materials into the cellular genome is not a concern. These properties make SeV vectors very promising for application to gene therapy (CYTOPLASMIC GENE THERAPY) and vaccination via the expression of therapeutic genes and antigens. In order to establish such SeV system, we have taken the strategy to generate the gene(s)-deleted or modified SeV vectors. Thus, we have succeeded in the recovery in high titers of fusion (F) gene-deleted (SeV/|[Delta]|F), matrix (M) gene-deleted (SeV/|[Delta]|M), hemagglutinin-neuraminidase (HN) gene-deleted (SeV/|[Delta]|HN), both M and F genes-deleted (SeV/|[Delta]|M|[Delta]|F), all of the envelop-related genes-deleted (SeV/|[Delta]|M|[Delta]|F|[Delta]|HN) SeV vectors by using the packaging cell lines which express respective proteins of those deleted gene(s). All the vectors showed efficient infectivity and transgene expression in various types of cell lines and primary cells in vitro. F gene-deletion made SeV vector non-transmissible, M gene-deletion worked well on the vector to become incapable of formation of the particles from infected cells, and HN gene-deletion was expected to reduce the host immune response against SeV vector. From this set of vectors, an appropriate type of SeV vector can be selected depending on the characteristics of applied diseases or protocols. By these vector engineering, a great advance of the SeV vector system has been achieved from the view of cytopathy and immunogenicity originated from SeV. The cytopathic reaction of SeV/|[Delta]|F was reduced from that of transmissible wild type SeV, and the additional gene(s)-deletion(s) from SeV/|[Delta]|F made much further reduction. Because the HN protein that mediates the cell-attachment of SeV is known to be one of the major targets for host immune responsive machineries on SeV infection, the HN gene-deletion from the genome was expected to be one of the important ways to reduce the immune response against SeV vector. As expected, the production of neutralizing antibody was reduced in the case of SeV/|[Delta]|M|[Delta]|F|[Delta]|HN. In addition, no activity of neutralizing antibody was seen when SeV/|[Delta]|M|[Delta]|F|[Delta]|HN was trasduced ex vivo. In this case, anti-HN antibody was not detected in the serum. Therefore, the gene(s)-deletion from SeV genome is very effective to reduce both the immunogenic and cytopathic reaction of SeV vector. Additional introduction of amino acid mutations identified from SeV strains showing persistent infection with slight lowered transcription and replication was also effective for the reduction of cytopathic effect. These modifications of SeV vector would improve the safety and broaden the range of the clinical application of this vector.

Background: Sendai virus (SeV) is negative-stranded RNA virus, which is classified as a type I parainfluenza virus belonging to the family of Paramyxoviridae. SeV has been known as rodent respiratory virus, however it is considered to be non-pathogen for humans. Recently, efficient gene transduction into various types of cells and tissue has been reported. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. Previously we reported Ang1 gene therapy clearly promoted myocardial angiogenesis and prevented remodeling in a rat AMI model. In the present study, we created recombinant Sendai virus vector (SeVV) encoding human Ang1 and evaluated therapeutic effect in the cardiovascular diseases. We also evaluated the angiogenic effect of mesenchymal stem cells (MSCs) transplantation with SeVV-mediated Ang1 gene modification.

Although airways are important targets for gene therapy for diseases such as cystic fibrosis or asthma, few vectors can transduce these organs. Recombinant Sendai virus (SeV) vectors can easily penetrate the mucus layer and efficiently transduce airway epithelial cells from apical side. However, the expression of transgene by the vectors of present design is transient and generally lasts only for several days. To improve lentivirus entry into airway epithelial cells, we have attempted to make the novel pseudotyped Simian Immunodeficiency Virus (SIV) vector with SeV envelope proteins, F and HN. The SIV vector could not be pseudotyped with the intact F and HN, but could be successfully pseudotyped after modification of the cytoplasmic portion of both proteins (Kobayashi et al. ASGT 2002). The new F and HN pseudotyped SIV vector was produced to the titers of more than 106 TU/ml and could be easily concentrated by centrifugation. When 108TU of SIV pseudotyped with F and HN was administered to mouse lungs through "sniffing", distinct transgene (GFP) expression was detected in the alveolar epithelium (Figure 1) and bronchiolar epithelial cells (Figure 2) ten days after the transduction. In contrast, VSV-G pseudotyped SIV vectors did not transduce the lung. These results suggest that our novel pseudotyped SIV vector with F and HN may be of great value as a tool for gene therapy of respiratory diseases.

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative strand RNA belonging to Paramyxoviridae family. We have previously shown that recombinant SeV as well as its modified non-transmissible variants can efficiently transfer and express foreign genes for a broad range of mammalian cells and tissues in vitro and in vivo and are suitable for use in gene therapy. SeV has two envelope glycoproteins HN and F, which mediate the virus attachment to and penetration into target cells, respectively. These envelope glycoproteins are not only important factors for viral tropism but also major determinants of immunogenicity in infected animals. Here we report the development of a novel SeV RNA replicon vector system that takes advantage of SeV self-replicating ribonucleoprotein complexes (RNP). In the vector system, RNP itself is non-infectious and can be infectious by the addition of lipofection reagents. In this system, nuclear entry of SeV RNA genome which forms an RNP complex with SeV NP, P and L proteins is not required and high expression of transgene is expected in transduced cells by self-replication of RNP in the cytoplasm. In addition, no effect of transduction of transgene in the presence of neutral antibody against SeV is expected. As the source of RNP, non-infectious virus-like particles (VLP) were recovered by infecting F-defective SeV vector into LLC-MK2 cells and harvesting VLP from the supernatants of the infected cells. To qualitatively and quantitatively compare this new vector system with conventional SeV vector or plasmid DNA, we introduced β-galactosidase, luciferase or enhanced green fluorescent protein (EGFP) reporter gene into the F-defective SeV genome, and recovered VLP from cloned cDNAs. The SeV RNA replicon vector mixed with lipofection reagents efficiently transferred foreign genes to several mammalian cell lines in vitro, and dose-dependently expressed luciferase or EGFP genes in tranduced cells. The expression level of SeV RNA replicon vector was comparable to that of SeV F-defective vector and remarkably higher than plasmid DNA-liposome complexes. Transduction of a foreign gene by SeV RNA replicon vector was not inhibited by the addition of an anti-SeV-HN antibody, although transduction by conventional SeV vector was completely inhibited. Therefore, the SeV RNA replicon vector system is useful for cytoplasmic expression of foreign genes in vitro and may be of help to its application to human gene therapy such as repeated administration and targeting of the vector.

Top of pageAbstract Sendai virus (SeV) vector has been shown to transduce the airway epithelial cells very efficiently. This property makes the vector promising for cystic fibrosis (CF) gene therapy. Previously, we generated a series of SeV vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene and showed that SeV can mediate CFTR gene transfer both in vitro and in vivo. CFTR activity in such experiments was confirmed in C127 cells in vitro using a radioactive iodide efflux assay and a whole cell perforated patch-clamp assay, and in CF knockout mice in vivo. These results indicated that SeV vector was a good candidate vector for CF gene therapy. However, there was not clear correlation between the expression levels of CFTR estimated from the design of a series of the vectors and observed activities of them. Additionally, cytotoxicity was observed in cells transduced by some types of SeV vectors probably or largely because of the overexpression of CFTR. To clarify these matters, biochemical analysis for CFTR protein itself is indispensable because CFTR is known to be highly glycosylated and glycosylation is responsible for mature CFTR function. However, we could not detect the CFTR by Western blotting using anti-CFTR antibody in SeV vector-transduced cells. Thus, we constructed a recombinant SeV vector designated SeV(HNL)-GFPCFTR/ΔF in which the N-terminus of CFTR was fused with an useful tag, GFP gene. The fusion gene was introduced into the junction between the hemagglutinin-neuraminidase (HN) and large protein (L) genes on the F gene-deleted SeV (SeV/ΔF) genome. It has previously been shown that GFP fusion to the N-terminus of CFTR did not affect processing, localization and function of CFTR (Bryan D. et al. 1998, J Biol Chem 273, 21759-68). We characterized the GFP-CFTR in the cells transduced with SeV(HNL)-GFPCFTR/ΔF. Western blotting using an anti-GFP antibody detected two proteins at 190 kDa and 170 kDa. These correspond in size to fully glycosylated mature CFTR and to the core-glycosylated immature protein, respectively. The mature and immature CFTR are known to be distinguishable by their different sensitivity to glycosidases. As expected, the 190 kDa protein was sensitive only to PNGaseF glycosidase and 170 kDa protein was to both PNGaseF and EndoH glycosidase. These results demonstrate that glycosylation of GFP-CFTR fusion protein derived from SeV(HNL)-GFPCFTR/ΔF was quite similar to endogenous CFTR protein. The subcellular localization of GFP-CFTR in MDCK cells infected with SeV(HNL)-GFPCFTR/ΔF was further analyzed by confocal laser microscopy. The GFP-CFTR was found to predominantly localize to the apical membrane. Thus, the proper maturation of CFTR protein in SeV vector-transduced cells was proved, indicating again that SeV is useful for CF gene therapy. Additionally, the generated SeV(HNL)-GFPCFTR/ΔF would be an useful system to analyze dynamics of CFTR protein including the tracking both in vitro and in vivo. The quantitative and functional analyses are now under investigation.

We have described gene delivery in sheep as a large animal model for airway gene transfer. This model allows comparison of different GTAs in terms of efficacy and safety using doses, delivery methods and assays, relevant to human studies. Proof-of-principle for non-viral gene transfer has been established however, levels of transgene expression are too low on a per cell basis to characterise which cells are targeted. Our preferred method for delivery is by aerosol however, not all GTAs are stable following aerosolisation in conventional jet or ultrasonic nebulisers and the cost of producing GTAs in sufficient quantities for whole lung aerosol studies can be prohibitive. These issues were behind our initial studies with delivery by instillation to individual segments. Unfortunately this approach is associated with increased toxicity as a result of pooling of material in the terminal bronchiole/alveolar duct regions. The Trudell AeroProbe* catheter develops an aerosol at the tip and can be passed through a bronchoscope. This allows localised delivery of GTAs, reducing the amount of material required. A further advantage of the AeroProbe* is that it is a single-pass aerosol device and subjects the GTA to less shear stress. An important question to be addressed when comparing delivery with different devices, is how their respective distribution patterns compare. Here we used a recombinant Sendai virus (DF/SeV-LacZ) vector, shown to be highly efficient at transducing airway epithelial cells in a variety of species, to investigate distribution patterns of delivery methods. SeV was delivered either by instillation to single lung segments via a polyethylene catheter (PEC), by AeroProbe* directed at segments or by AeroProbe* directed at the whole lung. In terms of stability, 100% and 49 +/- 3% of infectious virus particles survived the PEC and AeroProbe* respectively (n=3/group). In vivo studies show the variation in distribution of the different delivery methods. Instillation of 5ml SeV (3e9 CIU) to the same segment of different animals (n=3) results in varied distribution. Data from these animals confirm the absence of spillover as determined by histological assessment of adjacent segments. Segmentally-directed delivery of 5ml SeV (1e10 CIU) via AeroProbe* gives a different distribution. We observed areas of expression at airway bifurcations from impaction of aerosol particles at these sites and expression around sub-mucosal gland openings and within the glands themselves. Spillover to adjacent segments was observed in this case. Delivery of 24ml SeV (4e10 CIU) to the whole lung via the AeroProbe* gave a different type of expression pattern again. SeV was distributed throughout the lung. An even pattern of distribution all the way to the peripheral alveolar regions can be observed in several segments although the extent varies. These distribution patterns will have implications for sampling. It is clear from these studies that the properties of SeV make it an ideal vector to study the distribution pattern of different devices in our ovine model.

Background: Sendai virus (SeV), a rodent respiratory virus considered non-pathogenic for humans, is a negative-strand RNA virus, classified as a type I parainfluenza virus belonging to the family Paramyxoviridae. Recently, efficient gene transduction into various primary cultured cells and tissues by a recombinant Sendai virus vector (SeVV) has been reported. However, the feasibility of using SeVV in the context of myocardial gene therapy was not evaluated. In the present study, we evaluated SeVV-mediated gene-transfer into cardiomyocytes in vitro and in vivo