Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers.

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.

Modular retro-vectors for transgenic and therapeutic use.

Retroviruses have been used for many years as vectors for human gene therapy as well as for making transgenic animals. However, the efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and by the production of replication-competent retroviruses (RCR). Solutions to these and other difficulties are being found in modular vectors, in which the desirable features of different vector systems are combined. Examples of synergistic vectors include virosomes (liposome/virus delivery), adeno-retro vectors, and MLV/VL30 chimeras. As gene delivery systems become increasingly complex, methodology is also needed for precise assembly of modular vectors. Gene self-assembly (GENSA) technology permits seamless vector construction and simultaneous, multifragment assembly.

Fusogenic effects of murine retroviruses and cationic enhancers of transduction.

The maturation of retrovirus particles involves proteolytic cleavage of the envelope glycoprotein transmembrane component, resulting in conversion of the virus particle to a fusogenic or infectious state. Susceptible murine cells exposed to virus-containing supernatants from ecotropic retroviral helper cells occasionally fused to neighboring cells, resulting in syncytia (giant cells with multiple nuclei). Polycationic molecules dramatically enhanced the effect, leading to widespread cell death. The degree of cell fusion was dependent upon the retroviral envelope subtype (ecotropic-->amphotropic, gibbon ape leukemia virus was negative) as well as on the polycationic reagent used (G9 dendrimer-->Lipofectamine-->polybrene). Cell fusion effects were not mediated by the retroviral vector backbone, because virus-containing supernatants from helper cells (without vector) and vector producer cells had a similar effect. Human target cells were not fused by any type of murine retrovirus; in addition, amphotropic virus from human helper cells was not fusogenic toward murine cells. Thus, fusogenic effects were important during the propagation of vectors using murine helper cells but were not a significant factor during the transduction of human cells.

Role of far upstream repressor elements controlling proto-Ha-ras gene transcription.

The far upstream region of the rat Ha-ras gene has been characterized to determine whether possible repressor sequences may control the low level of Ha-ras gene transcription from its TATA-less, GC-rich strong promoter. The chloramphenicol acetyl transferase (CAT) gene under the control of the 3.8-kb Ha-ras upstream promoter was minimally expressed in HeLa cells. Surprisingly, CAT gene expression was increased by the deletion of a 0.7-kb BglII fragment containing non-coding exon minus 2 and TATA box promoter elements located 1.7 kb upstream of the GC-rich strong promoter. Far upstream (CA)25 repeats also appeared to repress Ha-ras gene activity. Sequences within the 0.7-kb BglII fragment suppressed CAT gene expression when placed upstream of a heterologous thymidine kinase (tk) gene promoter. Repressor activity was further localized to a 160-bp AvrII-BglII sub-fragment. Gel shift assays identified two sequence-specific DNA binding proteins. The results demonstrated for the first time that far upstream repressor sequences control normal transcription of the Ha-ras proto-oncogene.

Biosynthetic retrovectoring systems for gene therapy.

Chemical and physical methods of introducing genes into cells (transfection) are being combined with viral transduction as one possible approach toward overcoming the shortcomings of current gene transfer methods. Although several different strategies are being developed worldwide, this article focuses on modification of retroviral vectoring systems. One goal of this work is to combine the safety and ease of transfection methods with the permanency that is presently achieved only by integrating viruses. Work with retrotransposon pseudotypes, synthetic retrovectors, and liposomal delivery of retrovirus vectors is discussed.

Virosomes: cationic liposomes enhance retroviral transduction.

Retrovirus-derived vectors are overwhelmingly preferred over other methods for ex vivo gene therapy because they provide permanent integration of foreign genes into cellular DNA. In comparison, cationic lipids mediate efficent gene transfer, but expression is transient. When we combined cationic lipids with retrovirus particles we obtained a significant enhancement of transduction efficiency, depending upon the type of lipid formulation and the dose used. The relative effectiveness of these cytofectins was: DOSPA:DOPE > DOTMA:DOPE > DOTAP, resulting in 60-, 37-, and 5-fold increases in transduction efficiency, respectively, at optimum dosage. The effect of polycationic DOSPA:DOPE was dependent upon the viral envelope glycoprotein, was attainable by lipid treatment of either cells or virus particles, was not enhanced by the addition of polybrene, and was inhibited by chloroquine. These results strongly suggested that DOSPA:DOPE act primarily by modulation of charge associated with the viral envelope and cell membrane, enhancing retroviral transduction, rather than by providing an alternative pathway of transfection. DOSPA:DOPE is useful for improving the efficiency of gene transfer as well as the sensitivity with which retroviruses can be detected in biological fluids.

Expression of VL30 vectors in human cells that are targets for gene therapy.

Novel vectors made from mouse VL30 retrotransposons were tested in cell types that are targets for gene therapy, including normal human cells (skeletal muscle epithelium, bronchial epithelium, mammary epithelium), Epstein-Barr virus-transformed peripheral blood lymphocytes (PBLs), and various tumor cell lines. The long terminal repeat (LTR) transcriptional promoter, derived from the retroelement NVL-3, expressed abundant mRNA containing the bacterial neomycin resistance gene (neo) in all cell types tested. The amounts of neo RNA detected on RNA blots from normal vs. transformed cells were similar, although relatively less RNA was expressed in PBLs than in other cell types. Vector RNA expression in PBLs persisted during six months of continuous culture. Transcription was regulated by fibroblast growth factor (bFGF) and insulin, with the effects of each being cell-type-dependent. Thus, VL30 vectors introduced and expressed transgenes at significant levels in a number of cell types that are of interest for human gene therapy of metabolic and neoplastic diseases.

Transmission of endogenous VL30 retrotransposons by helper cells used in gene therapy.

Retrovirus-derived vectors and packaging cell lines are basic components used for gene transfer in human gene therapy. To eliminate recombinational and transcriptional problems associated with retroviral vectors, synthetic retrotransposon VL30 vectors were devised. During experimentation with these new vectors, extensive cotransmission of endogenous VL30 retrotransposon sequences was observed, originating within helper cell lines used in gene therapy experiments. The RNA was efficiently packaged into helper virus and was transmitted to recipient human cells, where it was again expressed as RNA. Transmission occurred regardless of whether the vector was retrovirus-derived or VL30, or if no vector was used. Endogenous VL30 RNA was readily detected in unselected recipient cells after a single exposure to helper virus, demonstrating a high efficiency of transmission compared with a cotransmitted VL30 vector, which contained an extensive deletion of internal sequences. These studies indicated that VL30 retrotransposons were ubiquitously transmitted by murine helper cells. Furthermore, the data strongly suggested that improvements in gene transfer may be obtained, both by using nonmurine helper cells (to reduce competitive inhibition by endogenous VL30) and by using VL30-derived vectors with intact packaging sequences.

Synthetic retrotransposon vectors for gene therapy.

New gene therapy methods are rapidly being developed to permit the expression of tumor suppressor genes, cytotoxins, anticancer antigens, and immunoregulatory proteins in the treatment of cancer. Large-scale testing in humans has been delayed by questions concerning the safety and effectiveness of preferred retroviral vectors and helper cells. These vector systems are limited by their ability to undergo homologous recombination with endogenous retroviruses or helper-viral sequences, resulting in release of replication-competent retrovirus (RCR). In addition, transcriptional inactivation of the retroviral promoter often occurs, caused in part by methylation of CpG islands in the retroviral long terminal repeats (LTRs). We report the production of highly specific retrovectors using gene amplification together with oligonucleotide building blocks. The synthetic vectors were based on mouse VL30 retrotransposon NVL3, and lacked homology to retroviral helper gene sequences. Three of four constructs made by gene amplification yielded biologically active vectors. These constructs efficiently transmitted and stably inserted a neomycin resistance marker gene into the genome of recipient cells, expressing an abundant RNA species of the expected size in the absence of detectable replication competent retrovirus. The vectors and techniques described enable widely applicable expression modes using generic helper cells, and require only approximately 1.3 kb of cis-acting vector RNA sequences for faithful transfer and expression of genetic material.

RB1 protein in normal and malignant human colorectal tissue and colon cancer cell lines.

The pattern of human retinoblastoma (RB1) gene protein expression was directly examined in normal and malignant human colorectal tissues and in seven colorectal carcinoma cell lines by immunohistochemistry using the mouse monoclonal antibody (RB-MAb-1) directed against the retinoblastoma protein (RB). This is the first demonstration of RB immunostaining in adult human colonic epithelium and colorectal carcinomas. Specificity using RB-MAb-1 was confirmed by western blot analysis, which showed bands of 110-116 kDa corresponding to the sizes of unphosphorylated and phosphorylated RB. RB staining of normal adult colonic epithelium was confined to the nucleus and was most intense in the transitional zone of the crypt, whereas lumenal cells (fully differentiated) were RB negative. Primary colorectal carcinomas and all the colon cancer cell lines stained positively for nuclear RB, but the expression was heterogeneous with varying fractions of RB negative cells present. Because we and others have previously shown that loss or inactivation of the RB1 gene is infrequent in colorectal carcinomas, reduced RB expression in such cells is probably due to a cellular regulatory mechanism. For example, RB negative cells may be those in early-G1 phase (known to have reduced RB levels) or growth-arrested cells that have differentiated. The ability to directly detect RB in primary colorectal carcinomas will permit assessment of whether heterogeneous expression of the RB1 gene product has prognostic significance for survival of patients with this cancer.

Transforming growth factor-alpha production and autoinduction in a colorectal carcinoma cell line (DiFi) with an amplified epidermal growth factor receptor gene.

The DiFi colorectal carcinoma cell line, derived from a patient with familial adenomatous polyposis, was examined for gene expression and production of the autocrine growth factor transforming growth factor alpha (TGF-alpha) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-alpha transcripts as identified on Northern (RNA) blots. Addition of TGF-alpha (10 ng/ml) or EGF (10 ng/ml) to DiFi cell cultures (lacking EGF or serum) up-regulated DiFi cell basal TGF-alpha mRNA levels, suggesting that autoinduction of TGF-alpha occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-alpha (347 pg/10(6) cells/24 h) into their culture medium, as determined by radioimmunoassay. DiFi cells showed strong overexpression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed enhanced EGFR expression in a cell subpopulation among the original (uncultured) ascitic fluid cells from which the DiFi cell line was established. This cell subpopulation was observed to expand dramatically between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphorylate. The EGFR gene in these cells was not found to be rearranged or genetically altered using Southern blot analysis. Dot blot analysis of DiFi cell DNA revealed EGFR gene amplification in the range of 60-80 copies/cell, which is approximately twice the copy number seen in A-431 epidermoid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because DiFi colorectal cancer cells uniquely show production and auto-induction of TGF-alpha in addition to amplification and overexpression of the EGFR gene, these cells represent a valuable tool for studying the role(s) of the EGFR in the regulation of tumor cell growth.

Liver-specific expression of a phosphoenolpyruvate carboxykinase-neo gene in genetically modified chickens.

In order to investigate the potential of the avian liver for the expression of recombinant proteins in vivo, replication-competent retroviral vectors were used to introduce a recombinant rat phosphoenolpyruvate carboxykinase promoter-driven neomycin resistance gene (PEPCKneo) into early Line 11 Leghorn embryos. After hatching, these birds possessed apparently intact PEPCKneo sequences in most tissues examined, however, the neo protein was expressed preferentially in the liver (up to .45% of total cellular protein). Therefore, the tissue specificity of the PEPCK promoter from the rat was retained in the chicken, although hormone responsiveness was not observed. Retroviral vectors used to transmit the genes were more stable during passage in either fibroblast cells or in the animal if the inserted genes were oriented in the same (sense) direction as the viral genome. After Geneticin drug selection in cultured cells, PEPCKneo mRNA was the predominant recombinant species observed on Northern blots, whereas embryos expressed mostly the RNA species originating in the retroviral long terminal repeats. The results demonstrate the potential usefulness of liver-specific gene expression in chickens, as well as the transcriptional effects observed when a foreign promoter is introduced into the replication-competent vector.

Casein, actin, and tubulin expression during early involution in bovine and murine mammary tissue.

Hybridization methods and in vitro translation were used to examine the expression and functional condition of messenger RNA encoding caseins and cytoskeletal proteins in the mammary gland during early involution. In the mouse, steady state mRNA levels for alpha-, beta-, and gamma-caseins coordinately decreased to 20% of initial levels between 12 and 72 h after pup removal. In vitro translatability of mouse casein mRNA, as determined by immunoprecipitation, electrophoresis, and gel slice counting, revealed a pattern that closely paralleled mRNA expression. In contrast, bovine casein mRNA levels were only slightly reduced by 72 h postmilking, whereas in vitro translatability decreased by about one-half. Northern blot analysis of total mouse mammary RNA that were hybridized with probes to cytoskeletal proteins showed a gradual decrease of alpha-tubulin mRNA, but an increase in beta-actin mRNA during early involution. Two-dimensional gel analysis of in vitro translated products indicated a concordant increase in beta-gamma-actin. In the cow, beta-actin mRNA at 72 h of involution was equal to or greater than that during lactation. These results demonstrate the generally slower involution response in the cow and suggest that differing regulations are involved. Early events of cellular involution may be related to a reorganization of the cytoskeleton.

Retrotransposon gene engineering.

We have used a mobile mouse VL30 genetic element together with retroviral helper cells to efficiently transmit and express chimeric foreign gene sequences in murine and human cells. The construct comprised a cDNA copy of retrotransposon NVL3, an internal promoter [rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32)] and an expressed bacterial neomycin resistance gene. Thirty to sixty thousand colony forming units/ml (CFU/ml) were recovered from the supernatant of mass cultured psi2 helper cells transfected with the recombinant retrotransposon plasmid DNA. RNA was expressed from both the VL30 long terminal repeat and from the internal PEPCK promoter, resulting in a G418 drug resistance phenotype in recipient cells. Integrated VL30 DNA sequences transduced from psi2 or PA317 retroviral helper cells failed to regenerate detectable replication competent virus. Human and rodent recipient cells transduced by the retrotransposons appeared to bear intact vector sequences after two rounds of transmission by helper cells.

Uptake and deoxyribonuclease 1 susceptibility of foreign deoxyribonucleic acid by chicken sperm cells.

Association of foreign DNA with chicken sperm cells was investigated using a 32P-oligolabelled plasmid preparation. Significant, although low, levels of trichloroacetic acid precipitable radioactivity became rapidly associated with viable sperm cells, in contrast to the situation with formaldehyde-fixed cells. However, analysis by Southern blotting and hybridization of sperm cells incubated with an unlabelled plasmid preparation followed by deoxyribonuclease 1 treatment, demonstrated associated foreign DNA was completely susceptible to nuclease degradation, whereas chromosomal DNA was not affected.

Efficient packaging of a specific VL30 retroelement by psi 2 cells which produce MoMLV recombinant retroviruses.

FTO-2B rat hepatoma cells acquired mouse VL30 retrotransposon(s) when infected with Moloney murine leukemia virus (MoMLV) recombinant retroviruses produced from psi 2 cells. The VL30 provirus was integrated into the rat genome, expressed at high levels, and its transcription induced 40-fold by dexamethasone, VL30 RNA was detected in hepatoma cells even without selection for the expression of the amino-3'-glycosyl phosphotransferase (neo) gene, which was co-transferred with a MoMLV retrovirus. However, the extent of transfer of the VL30 RNA was inversely related to the titer of the MoMLV recombinant retrovirus. The restriction map analysis of the transferred VL30 provirus was identical to the mouse VL30s of the NVL subfamily which is known to be a significant fraction of the transcriptionally active VL30 subset. Additionally, the regenerating liver from an adult rat, which was infected with a defective MoMLV-derived retrovirus, expressed VL30 RNA. These results indicate that great care should be given to the transfer of unwanted passengers, like VL30, present in retroviral packaging cell lines like the psi 2 cells, which are currently being used for gene therapy.