Search

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration. [ABSTRACT FROM AUTHOR]

We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/ Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors. [ABSTRACT FROM AUTHOR]

Abstract: While characterizing various splice forms of p120 catenin, we observed what appeared to be a novel posttranslational modification of p120, resulting in a higher molecular weight form that was dependent on the splicing pattern. Further investigation revealed the higher molecular weight form to be a fusion protein between sequences encoded by the retroviral vector and p120. We found that the publicly available sequence of the vector we used does not agree with the experimental sequence. We caution other investigators to be aware of this potential artifact. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events. [Copyright &y& Elsevier]

Abstract: Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however low vector concentration in batch cultures and high temperature-dependent decay limit sufficiently large-scale production. The ability of commercial ceramic asymmetric ultrafiltration membranes with a cut-off of 20 kDa to purify retroviral pseudotype vectors derived from the murine leukemia virus (MLV) carrying the HIV-1 envelope protein MLV (HIV-1) was studied. Experimental study was carried out in order to analyse the impact of inline tangential flow filtration (TFF) combined with different harvest temperatures in continuous production mode compared to batch filtration. Retroviral pseudotype vectors were produced using a 200 ml fixed bed reactor for high cell density cultivation on macro porous carriers up to 400 hours. By tangential flow ultrafiltration combined with cooling down the harvest supernatant to 4°C, the vector concentration was increased 12-fold with an average recovery of 78.7% of the initial infective capacity. [Copyright &y& Elsevier]

Abstract: Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-β SAR and/or 5′HS4 β-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5′HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5′HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects. [Copyright &y& Elsevier]

ABSTRACT The process of RNA interference (RNA1) has been exploited in cultured chicken cells and in chick embryos to assess the effect of specific gene inhibition on phenotypes related to development and disease. We previously demonstrated that avian leukosis virusbased retroviral vectors are capable of delivering effective RNAi against Marek's disease virus (MDV) in cell culture. In this study, similar RNAi vectors are shown to reduce the replication of MDV in live chickens. Retroviral vectors were introduced into d 0 chick embryos, followed by incubation until hatching. Chicks were challenged with 500 pfu of strain 648A MDV at day of hatch, followed by assays for virernia at 14 d postinfection. Birds were monitored for signs of Marek's disease for 8 wk. A stem-loop PCR, assay was developed to measure siRNA expression levels in birds. Delivery of RNAi co-targeting the MDV gB glycoprotein gene and ICP4 transcriptional regulatory gene significantly reduced MDV viremia in vivo, although to lesser extents than were observed in cell culture. Concomitant reductions in disease incidence also were observed, and the extent of this effect depended on the potency of the MDV challenge virus inoculum. Successful modification of phenotypic traits in live birds with retroviral RNAi vectors opens up the possibility that such approaches could be used to alter the expression of candidate genes hypothesized to influence a variety of quantitative traits including disease susceptibility. [ABSTRACT FROM AUTHOR]

Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of hematopoietic stem/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen has been a crucial factor in achieving stable engrafment of hematopoietic stem cells and therapeutic levels of ADA-expressing cells. Recent studies have demonstrated that gene therapy for ADA-SCID has favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Stem cell gene therapy combined with appropriate conditioning regimens might be extended to other genetic disorders of the hematopoietic system. [ABSTRACT FROM AUTHOR]

Abstract: Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl-beta-cyclodextrin (MbCD)) on the production of Molony murine leukemia virus (MoMuLV) retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than 2-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors. [Copyright &y& Elsevier]

Abstract: Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n =32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely. [Copyright &y& Elsevier]

Abstract: Genetically manipulated chickens producing chimeric monoclonal antibodies were generated by injecting retroviral vectors encoding genes for the heavy and light chains of antibodies into developing embryos. The transgene was detected in all chickens that hatched, and they stably produced the chimeric antibodies in their serum. After sexual maturation, the antibodies were also produced in eggs laid by the manipulated hens. The stable antibody production was observed both in egg white and yolk throughout the breeding period. The chimeric antibodies produced by the chickens were properly assembled and exhibited antigen-binding activities. Furthermore, we characterized the structures of the N-linked oligosaccharide chains added to the Fc-region of the recombinant antibodies produced in the serum, egg white and yolk of the chickens. [Copyright &y& Elsevier]

The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for α and β chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCR-transduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2′-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCR-based gene therapies targeting common epithelial malignancies. [ABSTRACT FROM AUTHOR]

A retroviral vector was injected into the heart of developing chicken embryos to determine the best timing for effective gene transduction into the gonads. Transduction efficiency and transgene expression were both highest when the viral solution was injected into embryos at stages 14–15 (Hamburger-Hamilton stages), in which primordial germ cells (PGCs) can be found in the blood circulation. This study shows that PGCs in the blood are important targets for gene transfer into the gonads using retroviral vectors. [Copyright &y& Elsevier]

Retroviral vectors are used in human gene therapy trials to stably introduce therapeutic genes in the genome of patients' cells. Their applicability, however, is frustrated by the limited viability of transformed cells and/or by risks linked to selection of oncogene-mutated clones. The reasons for these drawbacks are not yet completely understood. In this study, we show that LXSN-NeoR gene/interleukin-7-engineered mesenchymal stromal cells exhibited a marked enhancement of reactive oxygen species production compared with untransfected cells. This effect resulted to be independent on the product of the gene carried by the retroviral vehicle as it was reproducible in cells transfected with the empty vector alone. Stable transfection of mesenchymal stromal cells with the different retroviral vectors pBabe-puro and PINCO-puro and the lentiviral vector pSico PGK-puro caused similar redox imbalance, unveiling a phenomenon of more general impact. The enhanced production of reactive oxygen species over the basal level was attributable to mitochondrial dysfunction and brought back to altered activity of the NADH-CoQ oxidoreductase (complex I) of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with a cAMP analog, thus pointing to alteration in the protein kinase A-dependent signaling pathway of the host cell. Transfection of mesenchymal stromal cells with a PINCO-parental vector harboring the green fluorescent protein gene as selection marker in place of the puromyc-inresistance gene resulted in no alteration of the redox phenotype. These novel findings provide insights and caveats to the applicability of cell- or gene-based therapies and indicate possible intervention to improve them. [ABSTRACT FROM AUTHOR]

Abstract: Gammaretroviral and lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) have been used for stable gene transfer because of their broad host range and high mechanical strength. In the present study, an expression plasmid for chimeric VSV-G, consisting of a ZZ fragment derived from Staphylococcal protein A fused to the N-terminus of VSV-G (ZZ-VSV-G), was constructed to produce viral vectors capable of antibody-dependent gene transduction. Gammaretroviral (based on mouse stem cell virus, MSCV) and lentiviral (based on human immunodeficiency virus type 1, HIV-1) vectors pseudotyped with ZZ-VSV-G were produced without the loss of antibody-binding activity. The production of infectious viral particles was promoted by the addition of an expression plasmid for native VSV-G and antibody-dependent gene transduction was achieved using plates coated with antibodies. This system may be useful for the genetic transduction of cells expressing specific proteins on their surface, and for screening of antibodies specific for cell surface receptors. [Copyright &y& Elsevier]

Abstract: Tissue inhibitos of matrix metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs) and are associated with normal and pathologic extracellular matrix turnover. Because the microenvironment is critical for normal hematopoietic stem/progenitor cell function, we aimed to determine whether alterations in the TIMP/MMP balance impact upon normal hematopoiesis in mice. We have used both overexpression and knockout mouse models to determine whether early hematopoiesis is susceptible to potentially pathologic changes in TIMP/MMP level. These studies used TIMP-1−/− mice and retroviral vectors co-expressing human TIMP-1 or TIMP-2 linked with the green fluorescent protein (GFP) transduced into bone marrow (BM) cells and transplanted into lethally-irradiated recipient mice. Loss of TIMP-1 in knockout mice or retroviral overexpression of TIMP-1 or TIMP-2 did not alter hematopoietic stem/progenitor function during steady-state hematopoiesis. Surprisingly, even when applying hematopoietic stress through mobilization, chemotaxis, or myelosuppression, murine hematopoiesis was not adversely affected by TIMP-1 or TIMP-2 level. We conclude that TIMP/MMP balance alone does not exert significant influence on blood cell development and homeostasis. An important corollary of these studies is that specific modulation using MMP inhibitors for cancer or immunologic therapy is unlikely to have adverse hematopoietic side effects. [Copyright &y& Elsevier]

Abstract: RNA interference (RNAi) is a promising antiviral methodology. We recently demonstrated that retroviral vectors expressing short-hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) can be effective in reducing replication of other retroviruses in chicken cells. In this study, similar RNAi vectors are shown to inhibit replication of the avian herpesvirus, Marek''s disease virus (MDV, also known as gallid herpesvirus type 2), and its close relative, herpesvirus of turkeys (HVT). Cells expressing shRNA-mirs targeting the MDV or HVT gB glycoprotein gene or the ICP4 transcriptional regulatory gene show significant inhibition of viral replication. Not only are viral titers reduced, but observed plaque sizes are significantly smaller when the virus is grown on cells in which RNAi is effective. We also describe a modified retroviral delivery vector that expresses a shRNA-mir containing up to three RNAi target sequences and employ this vector with multiple targets within the MDV gB gene or within both the gB and ICP4 genes. The use of targets within multiple genes potentially can provide a larger antiviral effect and/or make it more difficult for viral escape mutations to evolve. [Copyright &y& Elsevier]

Abstract: The subventricular zone (SVZ) lining the lateral walls of the lateral ventricles is one of the major neurogenic areas in the postnatal brain. Precursor cells in the SVZ migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. Cell replacement strategies utilizing the recruitment of these endogenous progenitors and their progeny to different areas of the brain hold great promise for the future, but much research is needed in order to understand the sequence of molecular signals necessary to induce proliferation, migration and site-specific differentiation of these cells. In this study we show that the SVZ cells can be redirected from their normal migration route and directed towards other brain regions when they are infected with retroviruses encoding the developmentally important transcription factors Islet-1 and Neurogenin-2. After co-transduction with these transcription factors, transduced cells could be detected in several areas of the brain. When located in the striatum, the reprogrammed cells displayed neuroblast-like morphology. Once removed from the striatal parenchyma and allowed to further differentiation in vitro they developed into β-III-tubulin positive neurons. [Copyright &y& Elsevier]

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRα- and β-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-γ secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRα- and β-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function. [ABSTRACT FROM AUTHOR]

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34+ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34+ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34+ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19+ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the kappa/lambda chain on Pax5-transduced CD19+ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors. [ABSTRACT FROM AUTHOR]

Abstract: Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells. [Copyright &y& Elsevier]

Abstract: The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white–hEpo was comparable to that produced by recombinant CHO cells. [Copyright &y& Elsevier]

Abstract: Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3±0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7±0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3±6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7±14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling. [Copyright &y& Elsevier]

Background The purpose of this study was to construct an 'artificial beta cell' that can exhibit physiologic glucose-stimulated insulin secretion for the treatment of type 1 diabetes. Methods Retroviral vector containing the glucokinase (GK) gene and mutated human proinsulin (mhPINS) gene was constructed. HepG2 cells were first infected with recombinant retrovirus carrying the GK and mhPINS genes, then selectively cultured with G418 to obtain the positive clones. GK and mhPINS gene transcription and expression were identified by radioimmunity, Western blot and RT-PCR techniques. Finally, the dose-response effect of glucose on insulin secretion from those HepG2 cells that expressed both GK and mhPINS genes was tested with HepG2 cells that only expressed the mhPINS gene as a control. Results HepG2 cells with transferred GK and mhPINS genes were selectively cultured with G418 and the positive clones were obtained in 3 weeks. Four clones with GK and mhPINS gene expression were selected from 20 positive clones by radioimmunity and Western blot. We picked up one clone with a strong GK and mhPINS gene expression and named it clone Beta. In clone Beta, differences in insulin secretion at 0.5 and 0.75 mmol/L glucose concentrations were not significant (P>0.05) and differences in insulin secretion at 2.0, 3.0, 4.0, 5.0 and 6.0 mmol/L glucose concentrations were not significant (P>0.05), while there were significant differences in insulin secretion at other glucose concentrations(P<0.05). The artificial beta cell, clone Beta, obtained a glucose-stimulated insulin secretion with maximal insulin secretion at 1.75-2.00 mmol/L glucose concentrations. Discussion An artificial beta cell that exhibits glucose-stimulated insulin secretion can be constructed successfully. [ABSTRACT FROM AUTHOR]

We describe the construction of a large-scale, orderly assembly of mutant ES cells, generated with retroviral insertions and having mutational coverage in >90% of mouse genes. We also describe a method for isolating ES cell clones with mutations in specific genes of interest from this library. This approach, which combines saturating random mutagenesis with targeted selection of mutations in the genes of interest, was successfully applied to the gene families of G protein-coupled receptors (GPCRs) and nuclear receptors. Mutant mouse strains in 60 different GPCRs were generated. Applicability of the technique for the GPCR genes, which on average represent fairly small targets for insertional mutagenesis, indicates the general utility of our approach for the rest of the genome. The method also allows for increased scale and automation for the large-scale production of mutant mice, which could substantially expedite the functional characterization of the mouse genome. [ABSTRACT FROM AUTHOR]

Abstract: Enhancing DNA repair activity of hematopoietic cells by stably integrating gene vectors that express O6-methylguanine-DNA-methyltransferase (MGMT) is of major interest for innovative approaches in tumor chemotherapy and for the control of hematopoietic chimerism in the treatment of multiple other acquired or inherited disorders. Crucial determinants of this selection principle are the stringency of treatment with O6-alkylating agents and the level of transgenic MGMT expression. Attempts to generate clinically useful MGMT vectors focus on the design of potent expression cassettes, an important component of which is formed by enhancer sequences that are active in primitive as well as more differentiated hematopoietic cells. However, recent studies have revealed that vectors harboring strong enhancer sequences are more likely to induce adverse events related to insertional mutagenesis. Safety-improved vectors that maintain high levels of MGMT expression may be constructed based on the following principles: choice of enhancer–promoter sequences with relatively mild long-distance effects despite a high transcription rate, improved RNA processing (export, stability and translation), and protein design. The need for optimizing MGMT protein design is supported by recent observations suggesting that the P140K mutant of MGMT, developed to be resistant to inhibitors such as O6-benzylguanine, may confer a selective disadvantage when expressed at high levels. Here, we provide a review of the literature exploring MGMT expression vectors for bone marrow chemoprotection, and describe experimental evidence suggesting that high expression of MGMT P140K induces a selective disadvantage in the absence of alkylating agents. We conclude that the appropriate design of expression vectors and MGMT protein features will be crucial for the long-term prospects of this promising selection principle. [Copyright &y& Elsevier]

Abstract: Introduction: Gene therapy could prevent bleeding in hemophilia. However, antibodies could inhibit coagulation, while cytotoxic T lymphocytes could destroy modified cells. The immaturity of the newborn immune system might prevent these immune responses from occurring after neonatal gene therapy. Materials and methods: Newborn dogs, cats, or mice were injected intravenously with a retroviral vector expressing human Factor IX. Plasma was evaluated for antigen and anti-human Factor IX antibodies. Cytotoxic T lymphocyte responses were evaluated indirectly by analysis of retroviral vector RNA in liver. Lymphocytes were evaluated for cytokine secretion and the ability to suppress an immune response to human Factor IX in mice. Results and conclusions: Hemophilia B dogs that achieved 942±500 ng/ml (19% normal) or 5±0.4 ng/ml (0.1% normal) of human Factor IX in plasma only bled 0 or 1.2 times per year, respectively, and were tolerant to infusion of human Factor IX. Normal cats expressed human Factor IX at 118+/−29 ng/ml (2% normal) in plasma without antibody formation. However, plasma human Factor IX disappeared at late times in 1 of 4 cats, which was probably due to a cytotoxic T lymphocyte response that destroyed cells with high expression. C3H mice were tolerant to human Factor IX after neonatal gene therapy, which may involve clonal deletion of human Factor IX-responsive cells. These data demonstrate that neonatal gene therapy does not induce antibodies to human Factor IX in dogs, cats, or mice. The putative cytotoxic T lymphocyte response in one cat requires further study. [Copyright &y& Elsevier]

Abstract: Two of the most well-known genetic mechanisms in mammalian cells which control the susceptibility of cells to productive infection by retroviruses and lentiviruses rely on the cellular Fv1 and Ref1 restriction factors, which act, after viral entry, to prevent productive infection through their interactions with viral capsid (CA) sequences. While previous studies of Fv1 restriction involving N- and B-tropic murine leukemia viruses (MLVs) had demonstrated that the identity of a single amino acid residue at CA110 (arginine vs. glutamic acid) determines whether the resulting virus is N (arg) or B-tropic (glu), analogous studies of dual-tropic MLVs, such as Moloney MLV (Mo-MLV), have shown that additional residues other than CA110 are also involved in the specification of dual-tropic host range. Here we have further studied the CA determinants of Mo-MLV host range, with an emphasis on identifying additional CA residues and unique combinations of CA residues which differentially influence the ability of the resulting virus to infect murine and human cells. First, we show that CA82, a residue previously identified to affect the pattern of Fv1 restriction of different MLV viruses in murine cells, is a particularly strong potentiator of B-tropism in an Mo-MLV background carrying a glutamic acid residue at CA110 (A110E substitution), and that interestingly, different residues at CA82 lead to distinct patterns of restriction in human but not in murine cells. We also identify another CA residue, CA214, as a similarly potent potentiator of B-tropism, in the context of the A110E substitution. While another substitution at CA110, A110R, leads to strong potentiation of N-tropism in murine cells, in the absence of additional mutations, we found that A110R alone was not sufficient to confer appreciable restriction in Ref1-expressing cells, despite the fact that authentic N-MLV shows strong restriction in those cells. In conjunction with the A110R substitution, substitutions at CA82, but not at CA214, do lead to significant restriction in human cells, thus demonstrating a distinction between the interactions between those two determinants of restriction and CA110. Finally, using cell lines engineered to express the TRIM5α hu gene product, recently identified as the Ref1 restriction factor, and RNAi technology to knock-down expression of TRIM5αhu in human cells, we directly demonstrate that the unique patterns of restriction observed in human cells with the different mutants are consistent with a TRIM5αhu-mediated restriction. These studies shed further light on the complex determinants within the viral CA gene product which control the susceptibility of murine and human cells to retroviral infection. [Copyright &y& Elsevier]

Efficient ex vivo/ in vivo selection of genetically modified hematopoietic stem/progenitor cells (HPCs) and T lymphocytes could greatly improve several gene therapy strategies. We have previously reported that primary murine HPCs, transduced with a bicistronic retroviral vector, co-expressing the catalytic subunit of gamma-glutamylcysteine synthetase ( γ-GCSh) and eGFP, could be selected byl-buthionine- S, R-sulfoximine (BSO). Upon ex vivo transduction with a low, defined gene dosage and BSO selection, HPCs were able to repopulate the bone marrow of syngeneic myeloablated hosts, showing multi-lineage expression [ Hum Gene Ther, 16 (2005), 711]. We now provide ‘proof-of-principle’ that the same strategy can be applied to the gene therapy of graft-vs.-host disease (GVHD) subsequent to allogeneic bone marrow transplantation (ABMT), and of chromosome X-associated chronic granulomatous disease (CGD). Transfer of the herpes simplex virus-thymidine kinase (HSV-Tk) ‘suicide’ gene into donor T lymphocytes is a potential method to control GVHD after ABMT. However, an efficient selection system is required to eliminate non-HSV-Tk-expressing T lymphocytes before administration to the patient. We now report that, upon transduction with a retroviral vector, co-expressing γ-GCSh and eGFP, and subsequent selection by BSO, over 95% human T lymphocytes were found to express eGFP; moreover, upon transduction with a novel retroviral vector co-expressing γ-GCSh and HSV-Tk, and subsequent BSO treatment, over 95% of T lymphocytes could be eliminated by ganciclovir. The efficacy of the γ-GCSh-BSO selection strategy was then tested on an in vitro model of CGD. Upon transduction of gp91 (phox)-deficient PLBKO cells with a novel bicistronic retroviral vector co-expressing human gp91 (phox) and γ-GCSh, exposure to BSO for 48 h eliminated most non-transduced cells, resulting in selection of gp91 (phox)-expressing cells, and reconstitution of NADPH oxidase activity. [ABSTRACT FROM AUTHOR]

Abstract: Using a versatile and highly sensitive retroviral microarray, we have investigated particle preparations from three different human packaging cell lines harboring retroviral vector systems based on human immunodeficiency virus (HIV) and murine leukemia virus (MLV). 293Rev/Gag/Poli cells inducibly express high titers of HIV-derived particles for packaging of HIV vectors. The Phoenix-GP and the Anjou 65 cell lines constitutively express MLV vector particles. We compared the transcription profiles of human endogenous retroviruses (HERVs) in all cell lines with the HERV sequences present in the particles. In addition, the influence of the transfected vector plasmid on the copackaging of HERVs was investigated. All particle preparations showed a defined pattern of endogenous retroviral sequences that differed from the cellular HERV expression pattern. HERV transcripts were observed in the particle preparations independent of whether a vector construct was coexpressed or not. Furthermore, our results suggest that particle preparations are frequently contaminated by cellular vesicles (exosomes) containing cellular RNAs including HERV transcripts. [Copyright &y& Elsevier]

The transfer of T-cell receptor (TCR) genes into primary human T-cells to endow their specificity toward virus-infected and tumor cells is becoming an interesting tool for immunotherapy. TCR-modified T cells are mainly generated by retrovirus-mediated gene transfer. To produce TCR-retrovirus particles, fibroblast packaging cell lines are the most common tool. We constructed two packaging cell lines based on the human suspension T-cell lymphoma line Δβ-Jurkat, which lacks endogenous TCRβ-chains and is therefore unable to express CD3 complexes on the cell surface. After supply of gag-pol (murine leukemia virus (Mo-MLV)) and env (GALV or MLV-10A1) genes, a green fluorescent protein (GFP)-encoding retrovirus vector was transduced into both packaging cell clones, which then stably produced GFP-retroviruses with titers of up to 4 × 105 infectious particles (IP)/ml. After transfer of a TCRα/β-encoding retrovirus vector, Δβ-Jurkat/GALV and Δβ-Jurkat/10A1 cells expressed CD3 molecules on the cell surface. CD3-high expressing packaging cells were enriched by fluorescence-activated cell sorter sorting. In these cells, the CD3 expression level directly correlated with the titer of vector particles. TCR-retroviruses efficiently transduced human T-cell lines and primary T cells. In conclusion, the method allowed the fast and easy generation of high virus titer supernatants for TCR gene transfer.Gene Therapy (2007) 14, 595–603. doi:10.1038/sj.gt.3302906; published online 18 January 2007 [ABSTRACT FROM AUTHOR]

We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3±16.1, 46.6±5.8 and 93.7±7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.Cancer Gene Therapy (2007) 14, 279–286. doi:10.1038/sj.cgt.7701013; published online 12 January 2007 [ABSTRACT FROM AUTHOR]

Abstract: Mucopolysaccharidosis I (MPS I) due to deficient α-l-iduronidase (IDUA) activity results in accumulation of glycosaminoglycans in many cells. Gene therapy could program cells to secrete IDUA modified with mannose 6-phosphate (M6P), and enzyme could be taken up by other cells via the M6P receptor. We previously reported that newborn MPS I mice that were injected intravenously with 109 (high-dose) or 108 (low-dose) transducing units/kg of a retroviral vector (RV) expressing canine IDUA achieved stable levels of IDUA activity in serum and had reduced disease in heart, eye, ear, and bone in a dose-dependent fashion. However, the dose required for improvement in manifestations of disease in other organs was not reported. High-dose and low-dose RV mice with an average serum IDUA activity of 1037±90U/ml (471-fold normal) and 43±12U/ml (20-fold normal), respectively, had complete correction of biochemical and pathological evidence of disease in the liver, spleen, kidney, and small intestines. Although mice that received high-dose RV had complete correction of lysosomal storage in thymus, ovary, lung, and testis, correction in these organs was only partial for those that received low-dose RV. Storage in brain was almost completely corrected with high-dose RV, but was not improved with low-dose RV. The correction of disease in brain may be due to diffusion of enzyme from blood. We conclude that high-dose RV prevents biochemical and pathological manifestations of disease in all organs in MPS I mice including brain. [Copyright &y& Elsevier]

Abstract: The therapeutic efficacy of adoptively transferred cytotoxic T lymphocytes (CTL) has been demonstrated in clinical trials for the treatment of chronic myelogenous leukemia, cytomegalovirus-mediated disease, and Epstein-Barr virus-positive B cell lymphomas. It is however limited by the difficulty of generating sufficient amounts of CTLs in vitro, especially for the treatment of solid tumors. Recent gene therapy approaches, including two clinical trials, successfully apply genetic engineering of T cell specificity by T cell receptor (TCR) gene transfer. In this review we want to elucidate several principles of the redirection of T cell specificity. We cover basic aspects of retroviral gene transfer, regarding transduction efficacy and transgene expression levels. It was demonstrated that the number of TCR molecules on a T cell is important for its function. Therefore, an efficient transfer system that yields high transduction efficiency and strong and stable transgene expression is a prerequisite to achieve effector function by redirected T cells. Furthermore, we consider more recent aspects of T cell specificity engineering. These include the possibility of co-transferring coreceptors to create for example functional T helper cells by engrafting CD4+ T cells with a MHC class I restricted TCR and the CD8 coreceptor and vice versa. Also, risks related to the adoptive transfer of TCR gene-modified T cells and possible safety mechanisms are discussed. Finally, we summarize recent findings describing transferred TCRs capable of displacing endogenous TCRs from the cell surface. [Copyright &y& Elsevier]

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the β-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins. [Copyright &y& Elsevier]

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/106 cells/d) and mGH (11 μg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice ( lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase ( p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy. [ABSTRACT FROM AUTHOR]

Abstract: Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu, tat and rev originating from the pathogenic SHIV-89.6P. Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4×107 autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection. [Copyright &y& Elsevier]

Because of its easy accessibility, the skin is a very attractive target for gene therapy purposes. To study potential clinical applications in a preclinical setting, appropriate animal models are needed. Pig skin is very similar to human skin, and a variety of human diseases that are potentially amenable to gene therapy applications also occur in pigs. Only a few studies have analyzed the engraftment of transduced keratinocytes (KC) in pigs, however, with limited success. We describe a porcine model in which pig KC were transduced ex vivo with a retroviral vector encoding a marker gene and subsequently grafted onto the autologous host, utilizing a relatively simple grafting technique. Enhanced transduction efficiency was achieved by an optimized transduction protocol including centrifugation of the retroviral vector at a temperature of 32°C. Transduced KC were then seeded onto acellular dermis, forming a stratified epidermis. Grafting was performed by creating full thickness wounds and placing the skin graft onto the muscle fascia, covered by a protective skin flap for several days. Successful engraftment of transduced KC was demonstrated by immunohistochemistry of biopsies taken at different time points, showing transgene expression in 40–50% of grafted KC. After 4 weeks, KC expressing a foreign marker gene was lost, suggesting a transgene-specific immune response in the immunocompetent pigs and highlighting the potential problems for clinical gene therapy studies when transferring new genetic material into a patient. The model presented here may be used to examine applications of skin gene therapy, where retroviral vectors encoding endogenous pig genes will be expressed in the skin. [ABSTRACT FROM AUTHOR]

Abstract: To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours. [Copyright &y& Elsevier]

Abstract: The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level. [Copyright &y& Elsevier]

Abstract: Establishing the cell lineage relationships of cells during development allows insight into when and where developmental decisions are made. In the developing spinal cord, the origin and fate of radial glial has yet to be determined. One way in which to address this question is to transplant enriched populations of radial glia into the ventricular zone (VZ) region of host embryos to examine the lineage and differentiation pattern of these cells. An indirect selection procedure using immunomagnetic beads (Dynabeads; Dynal Biotech) was used here to isolate spinal cord radial glia. This negative immunoselection procedure resulted in a high yield of radial glia. A fluorescent cytoplasmic dye (Cell Tracker Green CMFDA) was used to label radial glia before transplantation. The role of radial glia as progenitor cells can also be examined using a green fluorescent protein (GFP)-expressing retroviral vector. The retroviral vector allows dividing cells in the VZ region of the spinal cord to be tracked by labelling them with GFP. Both techniques were utilised here to successfully label and examine embryonic spinal cord radial glia in vivo after a microinjection of either fluorescently labelled radial glia or replication-incompetent GFP-expressing retrovirus in utero. [Copyright &y& Elsevier]

The production of retroviral vectors using a transient expression system has been improved to obtain a high-titer virus preparation that is difficult to produce using packaging cell lines due to the cytotoxic or cytostatic effect of transgenes. Here, we used one such production method, the so-called Q-vector system, and examined its potential for virus production. The Q-vector system could produce a similar level of viral vectors compared with the packaging cell system but the production seemed to depend on the size and nature of transgenes. In the process of investigation of the quantitative difference in viral components between the transient expression system and the packaging cell system, we found that the Q-vector system could express higher amounts of viral RNA and proteins compared with the packaging cell system. However, this did not lead to a higher virus titer compared with that produced by the packaging cell system. This suggests that retroviral RNA transcribed from the plasmid in the transient system seemed to be used mainly for translation and only some of the RNA molecules were packaged in viral particles. [Copyright &y& Elsevier]

Abstract: Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human γc chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the γc chain, the cells were treated with ganciclovir (GCV). The γc chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the γc chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial. [Copyright &y& Elsevier]

Abstract: Oligodendrocytes have been considered to originate in a restricted ventricular zone of the ventral neural tube and to migrate and mature in their final targets. However, recent studies indicate that oligodendrocytes arise from multiple distinct dorsoventral origins. In this study, we investigate oligodendrocyte lineage cells in the embryonic optic tectum of chick, which develops from the dorsal region of the neural tube and invasion of optic tract. Oligodendrocyte precursor cells (OPCs) first appeared bilaterally on either side of the floor plate at E5. With further development, OPCs increased and spread laterally and dorsally to populate the optic tectum. At E7, OPCs appeared in another site along the ventral midline of the third ventricle, just dorsal to the optic chiasm. To examine the migration routes of these ventrally derived OPCs, we used DiI tracing in the organic culture and retinal denervation. Our results reveal that OPCs dispersed bilaterally along the optic tract and then migrated to the optic tectum in the stratum opticum (SO). In addition to these extrinsic OPCs, OPCs intrinsic to the tectal ventricle zone were identified at E14 using a combination of immunohistochemistry and retroviral mediated lineage tracing studies. These data support stage-specific dorsoventral origins and distribution of oligodendrocytes populating the optic tectum. [Copyright &y& Elsevier]

Abstract: The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors. [Copyright &y& Elsevier]

Abstract: Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (α3β3γ2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the α3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials. [Copyright &y& Elsevier]

Abstract: Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB) that results in accumulation of glycosaminoglycans in many organs. We have previously reported that neonatal intravenous injection of a gamma retroviral vector (RV) expressing canine GUSB resulted in transduction of hepatocytes, high levels of GUSB modified with mannose 6-phosphate in blood, and reduction in disease manifestations in the heart, bone, and eye. However, it was unclear if liver was the only site of expression, and the effect upon other organs was not assessed. We demonstrate here that blood cells from these RV-treated MPS VII dogs had substantial copies of RV DNA, and expressed the RNA at 2% of the level found in liver. Therefore, expression of GUSB in blood cells may synergize with uptake of GUSB from blood to reduce storage in organs. The RV-treated dogs had marked biochemical and pathological evidence of reduction in storage in liver, thymus, spleen, small intestines, and lung, and partial reduction of storage in kidney tubules. The brain had 6% of normal GUSB activity, and biochemical and pathological evidence of reduction in storage in neurons and other cell types. Thus, this neonatal gene therapy approach is effective and might be used in humans if it proves to be safe. Both secretion of enzyme into blood by hepatocytes, and expression in blood cells that migrate into organs, may contribute to correction of disease. [Copyright &y& Elsevier]

Gene therapy for advanced breast cancer is anticipated to be a useful therapeutic approach. Strategies in ongoing clinical protocols can be divided into four groups: (1) suppression of oncogenes or transfer of tumor-suppressor genes; (2) enhancement of immunological response; (3) transfer of suicide genes; (4) protection of bone marrow using drug resistance genes. We have started a clinical study of multidrug resistance ( MDRl) gene therapy. Advanced breast cancer patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation (PBSCT) with MDRl-transduced hematopoietic cells, and then were treated with docetaxel. Two patients have been treated so far, and in vivo enrichment of MDRl-transduced cells with docetaxel treatment has been seen. Both patients are in complete remission and had no apparent adverse effects from the MDRl gene transfer. [ABSTRACT FROM AUTHOR]