Diverse plasmid DNA vectors by directed molecular evolution of cytomegalovirus promoters

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.     

High-efficiency promoter-dependent transduction by adeno-associated virus type 6 vectors in mouse lung

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, beta-actin promoter and splice donor, and a beta-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, efficient transduction can be achieved.     

Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer

Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.     

Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells.

Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1alpha (EF1alpha) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1alpha promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1alpha promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity.     

Sustained expression after nonviral ocular gene transfer using mammalian promoters

The CMV promoter drives high transgene expression and is one of the most commonly used promoters for gene transfer. Tissue-specific mammalian promoters provide an alternative, and it would be useful to have a system to directly compare them to viral promoters free from potential confounding vector-related effects. In this study, we describe how electroporation after subretinal injection of plasmid DNA can be used to perform comparative quantitative analysis of promoter activities. Luciferase assay of eyecup homogenates was carried out after coinjection/electroporation of pGL2, a plasmid containing the promoter fragment of interest coupled to the firefly luciferase gene, and pRL-CMV, a plasmid containing the CMV promoter coupled to the Renilla luciferase gene for normalization. This technique was used to compare activity of different fragments of the 5'-upstream region of the vitelliform macular dystrophy 2 (VMD2) gene, which is selectively expressed in the retinal pigmented epithelial (RPE) cells, and results indicated positive regulatory elements between -104 and -154 bp and between -424 and -585 bp. Addition of a fragment from intron 1 reduced the activity of the -585/+38 bp fragment by 75%. Deletion analysis implicated a 342 bp region near the 5'-end of intron 1 in the repression. Results of transient transfections in two cell lines that constitutively express VMD2 were similar, and results in transgenic mice were consistent, providing validation for promoter analysis by in vivo electroporation. We then explored the time course of expression of the -585/+38 VMD2 promoter fragment and found that compared to cassettes driven by CMV or SV40 promoters, which showed peak luciferase activity on day 2 followed by a rapid decrease in activity, the VMD2 promoter fragment showed lower activity initially, but the activity was sustained for up to 56 days (longest time point measured). A promoter fragment from another RPE-specific gene, Rpe65, showed a similar pattern of sustained expression for at least 112 days. These data indicate that nonviral gene transfer can be used to quantitatively evaluate the activity of promoter fragments independent of influence from viral vectors. A potentially important finding using this new technique is the demonstration that relatively sustained passenger gene expression can be achieved with nonviral gene transfer using mammalian rather than viral promoters.     

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.     

Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature

Although the use of nonviral vectors for gene therapy offers distinct advantages including the lack of significant inflammatory and immune responses, the levels of expression in vivo remain much lower than those obtained with their viral counterparts. One reason for such low expression is that unlike many viruses, plasmids have not evolved mechanisms to target to the nucleus of the nondividing cell. In the absence of mitosis, plasmids are imported into the nucleus in a sequence-specific manner, and we have shown in cultured cells by transfection and microinjection experiments that the SV40 enhancer mediates plasmid nuclear import in all cell types tested (Dean et al., 1999, Exp Cell Res 253: 713-722). To test the effect of this import sequence on gene transfer in the intact animal, we have recently developed an electroporation method for DNA delivery to the intact mesenteric vasculature of the rat. Plasmids expressing luciferase or GFP from the CMV immediate-early promoter/enhancer and either containing or lacking the SV40 enhancer downstream of the reporter gene were transferred to the vasculature by electroporation. When transfected into actively dividing populations of smooth muscle or epithelial cells, the plasmids gave similar levels of expression. By contrast, the presence of the SV40 sequence greatly enhanced gene expression of both reporters in the target tissue. At 2 days post-transfer, plasmids with the SV40 sequence gave 10-fold higher levels of luciferase expression, and at 3 days the difference was over 40-fold. The presence of the SV40 sequence did not simply increase the rate of nuclear import and expression, since expression from the SV40-lacking plasmid did not increase beyond that seen at day 2, the time of maximum expression for either plasmid. In situ hybridization experiments confirmed that the increased gene transfer and expression was indeed due to increased nuclear localization of the delivered SV40 sequence-containing plasmid. Based on these findings, the ability to target DNA to the nucleus can increase gene transfer in vivo and inclusion of the SV40 sequence into plasmids will enhance nonviral gene delivery.     

Diverse stabilities of expression in the rat brain from different cellular promoters in a helper virus-free herpes simplex virus type 1 vector system.

Many neural gene transfer studies require both long-term and cell type-specific expression. We have reported a helper virus-free HSV-1 plasmid vector system (Fraefel et al., 1996), and this system supports at least some long-term expression from herpesvirus immediate-early promoters. In this study, we constructed vectors that placed the lacZ reporter gene under the regulation of five different cellular promoters. Vector stocks were microinjected into the midbrain, striatum, or hippocampus; the rats were sacrificed at 4 days to 2 months after gene transfer; and the numbers of X-Gal-positive cells were determined. A 6.8-kb fragment of the rat tyrosine hydroxylase (TH) promoter supported relatively stable expression for up to 2 months and targeted expression to TH-immunoreactive neurons in the substantia nigra pars compacta. The other promoters that were examined were chosen with the goal of obtaining long-term, neuronal-specific expression. At 4 days after gene transfer, a 766-bp fragment of the TH promoter supported expression in cells with neuronal morphology in the midbrain and striatum, consistent with results in transgenic mice. However, expression was absent by 2 weeks. Similarly, at 4 days after gene transfer, a mouse neurofilament heavy subunit promoter supported expression in cells with neuronal morphology in the midbrain, striatum, and hippocampus, but expression was absent by 2 weeks. A rat neuron-specific enolase promoter supported only a low level of expression in cultured neuronal cells, and expression was not detected in the brain. A rat voltage-gated sodium channel promoter supported only a low level of expression in PC12 cells and expression could not be detected in cultured cortical cells. These results demonstrate that different promoters support a wide range of levels and stabilities of expression in this vector system, and the results suggest approaches to improving the stability of long-term expression.