Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

Background

Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies.

Results

We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses.

Conclusions

RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.     

Plasmid vectors with multiple cloning sites and cat-reporter gene for promoter cloning and analysis in animal cells

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.     

Improved expression vectors for eukaryotic promoter/enhancer studies.

We describe two transfectable vectors designed to facilitate the functional analysis of eukaryotic promoter/enhancer sequences. The first, pJFCAT1, is an improved chloramphenicol acetyltransferase (CAT) reporter gene expression vector with two features that distinguish it from the majority of other CAT vectors currently in use: 1) it carries a trimer cassette of the simian virus 40 major late polyadenylation site to block plasmid-initiated read-through expression of CAT, and 2) it includes the phage f1 origin of replication, permitting generation of single-stranded copies to serve as templates for oligonucleotide-directed mutagenesis or single-strand DNA sequencing. The promoterless pJFCAT1 directs little if any CAT activity in transfected mouse L cells and, therefore, may be particularly useful for the analysis of weak promoters whose activity is otherwise masked by background CAT expression. The second vector, pTAG-1, uses human beta-globin as a reporter gene and was designed to facilitate the analysis of reporter gene expression at the RNA level. Like pJFCAT1, pTAG-1 also includes the simian virus 40 polyadenylation site trimer cassette located just upstream of the promoter insertion site. We have used each of these vectors to study functional elements in the human and mouse thymidine kinase promoters.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions.

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors

We previously reported that the HS-4 insulator, derived from the chicken beta-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an alpha-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.     

The Reliability of Marker/Reporter Genes in Biocontrol Studies with B. subtilis

A commercially available biocontrol agent, Bacillus subtilis MBI600, was transformed with plasmids containing a luciferase lux AB fusion cassette under the control of a constitutive promoter, as well as two promoter-less plasmids, containing either a lux AB fusion cassette or lux A and lux B genes. Introduction of these genes did not alter the growth rate, but subsequent expression of the luciferase enzyme resulted in a consistently compromised in vitro biocontrol activity. The expressed luciferase gene product (rather then the insertion of the novel genes) appeared to have undesired side effects on the phenotype of the transformed organism, demonstrating why caution should be exercised when using marker/reporter gene systems for investigating biocontrol.     

Construction of an adenovirus type 7a E1A- vector.

A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector.     

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.     

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice

It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland‐specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β‐casein, bovine αs1‐casein, or goat β‐lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β‐globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme‐linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17–8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7–40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland‐specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary‐specific expression of recombinant hLF in a transgenic mouse model.Mol. Reprod. Dev. 79: 573‐585, 2012. © 2012 Wiley Periodicals, Inc.     

Cooperative interactions between the GRP78 enhancer and promoter elements in hamster fibroblasts

A non-tissue-specific enhancer derived from the promoter of the rat 78-kDa glucose-regulated protein (GRP78)-coding gene was tested for its ability to stimulate the activity of its homologous promoter and two heterologous promoters (simian virus 40 and mouse mammary tumor virus). Single and double copies of the enhancer were inserted at positions 5' and 3' of the cat-expression vectors under the direction of the above promoters. The recombinant plasmids were transfected into hamster fibroblast K12 cells and assayed for chloramphenicol acetyl transferase activity under induced and non-induced conditions. We report that the GRP78 enhancer (i) exhibits strong cooperative interactions with its homologous promoter; (ii) can activate and confer a calcium ionophore (A23187) inducibility to heterologous promoters in an orientation-independent manner; (iii) prefers the 5' over the 3' location and; (iv) is dosage dependent in that two copies are twice as active as a single unit.