Improvement Of Transgene Expression Research Articles

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

BackgroundGenetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression.ResultsInitially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5–7.5% of transformants with 2–4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2–3-fold and 10–12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1–2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture.ConclusionsHere, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 ± 6% and by liposomal magnetofection by 85.1 ± 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R specific-shRNA by lipofection inhibited IGF-1R protein by an average of 43.8 ± 5.3%; that by liposomal magnetofection inhibited IGF-1R protein by 43.4 ± 5.7%, 56.3 ± 9.6%, and 72.2 ± 6.8%, at 24, 48, and 72 h, respectively, after pGFPshIGF-1R injection. Our findings indicate that liposomal magnetofection may be a promising method that allows the targeting of gene therapy to lung cancer.

Gene optimization mechanisms: A multi‐gene study reveals a high success rate of full‐length human proteins expressed in Escherichia coli

The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells.

Novel Cationic Lipids with Enhanced Gene Delivery and Antimicrobial Activity

Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D(2)S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D(2)S. D(2)S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at approximately 5 microM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D(2)S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D(2)S can improve transfection activity over common lipofection reagents, and D(2)S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation.

Gene transfer using self-assembled ternary complexes of cationic magnetic nanoparticles, plasmid DNA and cell-penetrating Tat peptide

Nonviral magnetofection facilitates gene transfer by using a magnetic field to concentrate magnetic nanoparticle-associated plasmid delivery vectors onto target cells. In light of the well-established effects of the Tat peptide, a cationic cell-penetrating peptide, that enhances the cytoplasmic delivery of a variety of cargos, we tested whether the combined use of magnetofection and Tat-mediated intracellular delivery would improve transfection efficiency. Through electrostatic interaction, gene transfer complexes were generated by mixing polyethylenimine-coated cationic magnetic iron beads with plasmid DNA, followed by addition of a bis(cysteinyl) histidine-rich Tat peptide. These ternary magnetofection complexes provided a 4-fold improvement in transgene expression at a dose of 1 μg of plasmid DNA per 20,000 cells over the binary complexes without the Tat peptide and transfected up to 60% of cells in vitro. The enhanced transfection efficiency was also observed in vivo in the rat spinal cord after lumbar intrathecal injection. Moreover, the injected ternary magnetofection complexes in the cerebrospinal fluid responded to a moving magnetic filed by shifting away from the injection site and mediating transgene expression in a remote region. Thus, our approach could potentially be useful for effective gene therapy treatments of localized diseases.

319. Improvement of Transgene Expression through the Direct Intrahepatic Injection of Adenoviral Vectors

Top of pageAbstract Numerous diseases could be potentially treated using gene therapy based strategies. An ideal gene therapy system should lead to a safe, low toxicity, long termed and high-level of expression of the therapeutic protein. However, the scaling up of the production for its application to general recombinant viral vector clinical treatment in humans presents a big problem for some of them, for example the third generation adenovirus. Focusing on the improvement of the high-level transgene expression of an adenoviral vector, we characterized a simple and efficient technique to transduce the liver.First and third generation adenovirus were used in mice to characterize the administration of the virus by direct intrahepatic injection. First and third generation adenovirus expressing human interleukin twelve (hIL12), as reporter gene, were assayed in mice by intravenous (IV) and intrahepatic (IH) administration. The expression of the transgene in the first generation adenovirus is under control of the CMV promoter. The third generation adenovirus, contains the hIL-12 gene under the control of an antiprogesterone receptor promoter system. In order to study the relevance of Kupffer cells in the adenovirus liver transduction, we injected both vectors after a clodronate containing liposomes injection. The IH injection of 1×1010 pfu of the first generation adenovirus Ad-IL-12 shows protein levels 2–3 times higher than the administration of the same dose by conventional IV administration. Il-12 expression it is not detected in mice receiving 109 pfu of the Ad-hIL-12 I.V., however the same dose administerred I.H. resulted in clearly detectable levels of the transgene in serum. That should result in an at least 2–3 times lower dose of virus needed to get an identical expression level. The previous injection of clodronate-liposomes improves substantially the expression in both routes and maintains the previously described effect. Moreover the kinetic of the transgene expression is different depending on the route of administration, after IH. administration the highest expression level of the transgene is detected at early time points and an abrupt progressive decrease, on the other hand adenovirus IV administerred showed the peak of transgene expression 48 hours after viral injection. The third generation adenovirus injected intrahepatically offers a better increase in protein expression, 3–4 times, respect to the IV injection. However the empty-liposome preinjection decreases this improvement and the clodronate-liposomes administration inverts this tendency, being the IV better than the IH, in this case. This method evades the phagocytosis by the liver resident macrophages and permits a good expression at low virus doses. Empty-liposomes poorly and the clodronate-liposomes clearly deplete the Kupffer cells and eliminate this difference. This novel technique of gene therapy administration offers several advantages. It makes feasible the administration of less adenovirus that expresses high levels of transgene. It presents a quick and easy treatment method for humans and superior mammals, especially for third generation adenovirus.

Nanoparticles of Compacted DNA Transfect Postmitotic Cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.

Coexpression of p21(WAF1/CIP1) in adenovirus vector transfected human primary hepatocytes prevents apoptosis resulting in improved transgene expression.

Replication-deficient adenovirus (Ad vector) is one of the most effective gene transfer systems. However, its employment in human gene therapy trials is hampered by Ad vector associated cytotoxicity and induction of apoptosis of the infected cells. Here, we identify one underlying mechanism as uncoupling of S phase and mitosis of the cell cycle leading to apoptosis and decline of transgene expression. Moreover, we demonstrate a strategy to avoid Ad vector associated cytotoxicity and induction of apoptosis in human primary hepatocytes by coinfection of Ad vector carrying the cDNA of choice and the cell cycle regulator p21(WAF1/CIP1) (p21). In addition, animal experiments were performed using Ad vector directed coexpression of p21 and human alpha 1-antitrypsin. As serum analysis of alpha 1-antitrypsin after Ad vector mediated gene transfer to the liver of mice revealed, this strategy resulted also in the improvement of transgene expression by two orders of magnitude. These data suggest that coexpression of p21 and Ad vector carrying a therapeutic gene may be a promising strategy to avoid cytotoxicity and induction of apoptosis leading to improved safety in human gene therapy.