Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.     

Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: a method to select producer cells and follow transduced target cells

The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones. When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.     

Use of amphotropic retroviral vectors for gene transfer in human colon carcinoma cells

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.     

Complementation of helper-dependent adenoviral vectors: size effects and titer fluctuations

The complementation of adenoviral vectors with large deletions in the viral genome was studied. The helper adenovirus used to complement these vectors contains a partial deletion of the packaging signal and the E1 region substituted by the lacZ gene. The effect of vector size on packaging efficiency was analysed in 293 cells using decreasingly shorter vectors expressing GFP from a CMV enhancer-beta-actin promoter. Vectors with longer genomes propagated more efficiently than shorter ones. Vectors containing only the packaging signal and the ITRs of Ad5, having all the viral genes replaced with unrelated sequences packaged as efficiently as vectors of the same size containing adenoviral DNA instead of exogenous DNA. The amounts of helper and vector produced in coinfected 293 cells exhibited the typical cycling fluctuation observed during serial propagation of a virus with defective interfering particles.     

Values and limits of adenoviral vectors for gene transfer in vivo

Review of the therapeutic use of DNA transfer to treat a certain number of hereditary diseases (such as adenosine deaminase deficiency) or acquired diseases. The strategies (ex vivo manipulation or direct in vivo transfer of the corrective gene), vectors (retrovirus, adenovirus, nonviral vectors), and diseases which can benefit from gene therapy are considered and discussed together with an evaluation of the risk of gene therapy.     

Gene therapy for malignant gliomas using replication incompetent retroviral and adenoviral vectors encoding the cytochrome P450 2B1 gene together with cyclophosphamide

Cyclophosphamide is an inactive prodrug which is converted by hepatic cytochrome P450 2B1 to cytotoxic metabolites which produce interstrand DNA cross-linking in a cell cycle-independent fashion. The limited ability of these metabolites to cross the blood-brain barrier contributes to the poor activity of cyclophosphamide against brain tumors. In this study we demonstrate that replication deficient retroviral and adenoviral vector-mediated gene transfer of cytochrome P450 2B1 into 9L glioma cells significantly increases the sensitivity of these tumor cells to cyclophosphamide in vitro, and prolongs the survival of animals bearing intracerebral 9L tumors treated with cyclophosphamide in vivo. Attempts to improve the effectiveness of retrovirally mediated transduction of the P450 2B1 gene by increasing the concentration of cyclophosphamide delivered to the tumors using intracarotid and intratumoral injections did not prolong animal survival, although survival was increased when a second treatment with P450-expressing retroviral vectors and cyclophosphamide was administered. These results suggest that in situ transduction of tumor cells with the P450 2B1 gene using retroviral and adenoviral vectors increases their sensitivity to cyclophosphamide and may have a potential role in the therapy of malignant gliomas.     

Fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells

T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).     

Use of the cosmid adenoviral vector cloning system for the in vitro construction of recombinant adenoviral vectors

The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation. Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro. The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors. To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors. In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells. The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy.     

Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo

OBJECTIVE: To determine whether individual surgeon experience is associated with improved short-term clinical and economic outcomes for patients with benign and malignant thyroid disease who underwent thyroid procedures in Maryland between 1991 and 1996. SUMMARY BACKGROUND DATA: There is a prevailing belief that surgeon experience affects patient outcomes in endocrine surgery, but there is a paucity of objective evidence outside of clinical series published by experienced surgeons that supports this view. METHODS: A cross-sectional analysis of all patients who underwent thyroidectomy in Maryland between 1991 and 1996 was conducted using a computerized statewide hospital discharge data base. Surgeons were categorized by volume of thyroidectomies over the 6-year study period: A (1 to 9 cases), B (10 to 29 cases), C (30 to 100 cases), and D (>100 cases). Multivariate regression was used to assess the relation between surgeon caseload and in-hospital complications, length of stay, and total hospital charges, adjusting for case mix and hospital volume. RESULTS: The highest-volume surgeons (group D) performed the greatest proportion of total thyroidectomies among the 5860 discharges, and they were more likely to operate on patients with cancer. After adjusting for case mix and hospital volume, highest-volume surgeons had the shortest length of stay (1.4 days vs. 1.7 days for groups B and C and 1.9 days for group A) and the lowest complication rate (5.1 % vs. 6.1% for groups B and C and 8.6% for group A). Length of stay and complications were more determined by surgeon experience than hospital volume, which had no consistent association with outcomes. CONCLUSIONS: Individual surgeon experience is significantly associated with complication rates and length of stay for thyroidectomy.     

A new retroviral vector for detecting mutations and chromosomal instability in mammalian cells

A retroviral vector carrying both forward (neo) and backward (herpes simplex virus thymidine kinase or HSV-TK gene) selection markers was constructed as a substrate for mutational assay in mammalian cells. The cells infected with this virus are first selected with G418, mutagenized and then selected with the anti-herpes drug acyclovir (ACV). Since HSV-TK, but not the host TK, is capable of converting ACV to a toxic metabolite, cells retaining the intact HSV-TK gene fail to survive, while the cells carrying a mutated HSV-TK gene or which have lost the gene can form colonies in the presence of ACV, making it possible to detect the genetic defects in a positive manner. It is also possible to discriminate between small mutations and large deletions by checking the presence of the linked marker, neo. As a model experiment, we prepared an uncloned pool of rat fibroblast cells (CREF) infected with this virus and irradiated them with increasing doses of ultraviolet light. Dose-dependent increases in the number of ACV-resistant colonies were observed. Structural analysis of the HSV-TK gene in these clones revealed point mutations or small deletions in the majority of the cases. Since it requires no pre-existing genetic markers in the host cells, this system may be used for a wide variety of mammalian cells and provides a useful tool to assess both their susceptibility to various mutagens and their genomic instability.     

Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection

Nine formulations were processed into bologna with different ratios of soy protein isolate (SPI):sodium caseinate (SCA), i.e. 1:1, 1:2.5, 1:5, 5:1, 5:2.5, 5:5, 10:1, 10:2.5 and 10:5. The products were evaluated for yields, emulsion stability, physical measurements (shearforce-kgf and folding test) and taste panel evaluation. Formulations with 5:1 and 5:5 SPI:SCA had lower liquid loss resulting in higher yields while the others had poor emulsion stability and high liquid loss. Firmer texture was exhibited by formulations 1:1, 5:1 and 10:1 SPI:SCA but formulation with 1:1 SPI:SCA showed better gelation followed by 1:2.5, 1:5, 5:1, and 5:2.5. The other formulations had poor gelation and binding properties, especially formulation with 10:5 SPI:SCA. Sensory evaluation was carried out using 30 untrained panelists. Attributes evaluated were aroma, texture, chewiness, juiciness, saltiness, chicken taste and overall acceptance. Formulation with 5:1 SPI:SCA was more acceptable for texture, chicken taste and overall acceptance while formulation with 1:1 SPI:SCA was more acceptable for the chewiness, juiciness and saltiness attributes. There was no significant difference (P > 0.05) in aroma attribute, for all formulations.     

FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells

FMEV retroviral vectors combine the long terminal repeat of Friend mink cell focus-forming viruses with the 5' untranslated leader region of the murine embryonic stem cells virus. These modules were connected to achieve high transgene expression in hematopoietic progenitor and stem cells. Here, we report the cloning of safety-improved and versatile FMEV vectors allowing module-wise exchange of crucial elements for comparative studies. By transfer and expression of four different marker genes (neomycin phosphotransferase, lacZ, enhanced green fluorescent protein and truncated low affinity nerve growth factor receptor), we formally demonstrate that both the long terminal repeat and the leader contribute to the high expression of FMEV in transduced hematopoietic cells. Most prominent are the data recorded in the absence of selection in myelo-erythroid progenitor cells. Here, FMEV vectors mediate up to two orders of magnitude increased transgene expression levels when compared with vectors based on the Moloney murine leukemia virus.     

Transfection of a retroviral construct carrying a non producer HIV-1 variant induces HIV-1 resistance in CD4+ CEMss cells

The phenotype of Human Immunodeficiency Virus-1 (HIV)-infected HUT-78 cell clone (F12) has been described (Federico et al, AIDS Res Hum Retrov 1989; 5: 365-96). Briefly, F12 cells are: i) CD4 down-regulated, ii) non producer and iii) fully resistant to homologous superinfection. We tested whether this phenotype was dependent upon the expression of the HIV-1 genome integrated therein. The SstI/SstI F12 provirus was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo)-Geneticine resistance gene. CD4+ HIV-susceptible CEMss cells were transfected with this construct in the sense orientation. Neo-resistant clones exhibited an integrated viral DNA, low viral mRNA expression and (as in F12 cells) the presence of uncleaved gp160, no gp41 and a small amount of p55 gag precursor. Superinfection of the F12/HIV-DNA-transfected CEMss clones showed that these CD4+ cells had acquired a significant (0.7-1.5 logs) resistance towards superinfection with HIV-1. This was observed in all four transfected clones where the F12/HIV DNA was expressed, but not in the control clone that was transfected with the pLj vector alone. These results confirm those that were obtained with human CD4+ CEMss cells infected with a recombinant retrovirus bearing the same SstI/SstI F12/HIV genome (Federico et al, J Gen Virol, 1993, in press). Both sets of results indicate that the expression of this genome in bio-engineered CD4+ human cells results in their intracellular immunization against HIV-1.     

Provision of positive and negative selections in retroviral vectors containing the cytosine deaminase gene

The E. coli cytosine deaminase (CD) provides a negative selection system for suicide gene therapy as CD transfectants are eliminated following 5-fluorocytosine (5FC) treatment. Here we report a positive selection system for the CD gene using 5-fluorouracil (5FU) and cytosine in selection medium to screen for CD-positive transfectants. It is based on the relief of 5FU toxicity by uracil which is converted from cytosine via CD catalysis, as uracil competes with the toxic 5FU in subsequent pyrimidine metabolism. Hence, a retroviral vector containing the CD gene may provide both positive and negative selections after gene transfer. The CD transfectants selected with the positive selection system showed susceptibility to 5FC in subsequent negative selection in vitro and in vivo. Therefore, this dual selection system is useful not only for combination therapy with transgene and CD gene, but can also act to eliminate selectively transduced cells after the transgene has furnished its effects or upon undesired conditions if 5FC is applied for negative selection in vivo.     

The selectable marker neo gene down-regulates gene expression from retroviral vectors containing an internal ribosome entry site

The internal ribosome entry site (IRES) from the picornavirus family has frequently been used to express multiple genes from a polycistronic message in retroviral vectors. While examining factors affecting levels of gene expression in IRES-containing retroviral vectors, it was found that retroviral vectors expressing the two genes linked by IRES, the reporter gene and the selectable marker neo, produced significantly lower levels of protein than those containing a reporter gene alone. This observation has been made with various cDNA sequences. However, when the neo was replaced with a different cDNA, the level of gene expression was increased, often to the level achieved with a vector expressing a single gene, suggesting that the bacterial neo sequence has a negative effect on expression. Analysis of the steady-state RNA levels isolated from transfected packaging cells showed that the neo-containing retroviral vectors produced significantly lower levels of RNA than those lacking this bacterial sequence indicating that neo interferes with expression of the neighboring gene at the level of RNA. Furthermore, the order of genes in the IRES-neo-containing vectors appeared to be more important than in the vector lacking the neo sequence. Our results suggest that neo has to be used in the retroviral vector with care, especially when a high level gene expression is needed.     

Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene

The purpose of this study was to determine whether retrovirus-mediated transfer of the murine macrophage inducible nitric oxide synthase (iNOS) gene can inhibit tumorigenicity and metastasis of human renal cancer cells. Retroviral vectors encoding murine macrophage iNOS were constructed in the pLXSN retroviral vector with the iNOS gene under the control of a long terminal repeat promoter and a neomycin resistance gene under the control of an internal simian virus 40 promoter. Highly metastatic human renal carcinoma SN12PM6 cells were infected with control or iNOS retrovirus. Expression of iNOS was confirmed by Northern and Western blot analyses, and expression of the functional iNOS protein, i.e., production of nitric oxide (NO), was determined by measuring nitrite accumulation in culture supernatants. Noninfected or control cells produced large orthotopic tumors in the kidney of nude mice and a larger number of experimental lung metastases, whereas iNOS-infected cells produced small tumors in the kidneys and few to no lung metastases. The data indicate that the infection of human renal cancer cells by retroviruses harboring the murine iNOS gene can induce the production of high levels of NO, which is associated with autocytotoxicity, suppression of tumorigenicity, and abrogation of metastasis.     

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line

Magnetic resonance imaging (MRI) was used in 13 patients with peripheral lymphedema and 2 patients with extensive cavernous lymphangioma of the limb for the purpose of evaluating its role in diagnosis of lymphatic disorders. In chronic lymphedema, MRI showed deformity of lymphatics at different tissue levels. In the subcutis, MRI characteristically displayed diffuse edema or a honeycombed pattern consistent with reticular lymphangiectasis and "lakes" with a marked increase in signal intensity with T2-weighted imaging. In lymphedema hyperplasia and chylous reflux, MRI depicted dilated retroperitoneal lymphatic collectors and lumbar trunks. In cavernous lymphangiomatosis, MRI demonstrated a prominent lattice-like pattern which had lower signal intensity on T1-weighted imaging and higher intensity on T2-weighted imaging. The findings of MRI are valuable not only for accurate assessment of lymphatic dysplasia syndromes but also provide a blueprint for treatment options.