Efficient Nonviral Gene Transfer Research Articles

DNA nuclear targeting sequences for enhanced non-viral gene transfer: An in vitro and in vivo study

An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NFs are optimized short nucleotide sequences able to interact with the transcription factor nuclear factor κB (NF-κB), which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNAs featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNAs were hydrodynamically delivered to muscle hindlimbs in healthy mice; this direct accurate comparative (in the absence of any gene carrier) revealed modest but consistent trends in favor of the pDNAs equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NF-κB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.

A highly soluble Sleeping Beauty transposase improves control of gene insertion.

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk for genome instability, and by the inability to use transposase protein directly. Here, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T-cells, which exhibit potent anti-tumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR-T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

Apoptin Gene Delivery by the Functionalized Polyamidoamine (PAMAM) Dendrimer Modified with Ornithine Induces Cell Death of HepG2 Cells.

The use of tumor-specific therapeutic agents is a promising option for efficient and safe nonviral gene transfer in gene therapy. In this study, we describe the efficacy of polyamidoamine (PAMAM)-based nonviral gene delivery carriers, namely, an ornithine conjugated PAMAM (PAMAM-O) dendrimer in delivering apoptin, a tumor-specific killer gene, into human hepatocellular carcinoma (HepG2 cells) and dermal fibroblasts. We analyzed the transfection efficiency by the luciferase assay and assessed cell viability in both cell types. The transfection efficiency of the PAMAM-O dendrimer was found to be higher than that of the PAMAM dendrimer. Moreover, the cytotoxicity of the PAMAM-O dendrimer was very low. We treated both cell types with a polyplex of PAMAM-O dendrimer with apoptin, and analyzed its cellular uptake and localization by confocal microscopy. Cell cycle distribution, tetramethylrhodamine, ethyl ester (TMRE) analysis, and transmission electron microscopy imaging showed that apoptin induced cell death in HepG2 cells. We therefore demonstrated that a PAMAM-O/apoptin polyplex can be used as an effective therapeutic strategy in cancer owing to its effectiveness as a suitable nonviral gene vector for gene therapy.

Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1.

Non-viral gene transfection in vitro using endosomal pH-sensitive reversibly hydrophobilized polyethylenimine

Reversibly hydrophobilized 10 kDa polyethylenimine (PEI) based on rapidly acid-degradable acetal-containing hydrophobe was designed for nontoxic and highly efficient non-viral gene transfer. Water soluble PEI derivatives with average 5, 9 and 14 units of pH-sensitive 2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME) hydrophobe per molecule, denoted as PEI- g-(TMB-THME) n, were readily obtained by treating 10 kDa PEI with varying amounts of TMB-THME-nitrophenyl chloroformate. Gel retardation assays showed that all PEI- g-(TMB-THME) n derivatives could effectively condense DNA at an N/P ratio of 5/1. Notably, polyplexes of PEI- g-(TMB-THME) n derivatives had smaller sizes (about 100∼170 nm) and higher surface charges (+25 ∼ +43 mV) than the parent 10 kDa PEI at the same N/P ratios ranging from 10/1 to 40/1. MTT assays revealed that these PEI- g-(TMB-THME) n derivatives were practically non-toxic at polymer concentrations used in transfection experiments. The acetal degradation of PEI- g-(TMB-THME) 9 was shown to be highly pH dependent in which half lives of 1.3, 2.8 and 11 h were determined for pH 4.0, 5.0 and 6.0, respectively, while negligible hydrolysis (<12%) was observed after 24 h at pH 7.4. Gel electrophoresis, dynamic light scattering (DLS) and zeta potential analyses indicated that polyplexes formed at an N/P ratio of 10/1 were dissociated following 5 h incubation at pH 5.0, highlighting the importance of hydrophobic TMB-THME moieties in DNA condensation and supporting that acetal hydrolysis in endosomes would facilitate DNA release. Notably, in vitro transfection experiments performed at N/P ratios of 10/1 and 20/1 in HeLa, 293T, HepG2 and KB cells using plasmid pGL3 expressing luciferase as the reporter gene showed that reversibly hydrophobilized PEIs had superior transfection activity to 25 kDa PEI control. For example, polyplexes of PEI- g-(TMB-THME) 14 showed about 235-fold and 175-fold higher transfection efficiency as compared to 10 kDa PEI in HeLa cells in serum-free and 10% serum media, respectively, which were approximately 7-fold and 16-fold higher than 25 kDa PEI formulation at its optimal N/P ratio under otherwise the same conditions. Confocal laser scanning microscope (CLSM) studies confirmed that PEI- g-(TMB-THME) 14 efficiently delivered Cy5-labeled DNA to the nuclei of HeLa cells. These endosomal pH-sensitive reversibly hydrophobilized PEIs have great potentials for safe and efficient non-viral gene transfection.

Branched Polyethylenimine Derivatives with Reductively Cleavable Periphery for Safe and Efficient In Vitro Gene Transfer

Twenty-five kDa polyethylenimine (PEI) is one of the most efficient nonviral gene transfer agents currently applied as a golden standard for in vitro transfection. In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to enhance further the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl-N'-acryloyl-cystamine (Ac-Cys-(t)Boc) and N-tert-butoxycarbonyl-N'-methacryloyl-cystamine (MAc-Cys-(t)Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)x(Ac) and PEI-(Cys)x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2 to 23.1% versus 15.1%). Gel retardation and ethidium bromide exclusion assays showed that cystamine modification resulted in largely enhanced interactions with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared with 25 kDa PEI with IC(50) values of >100 mg/L and 50-75 mg/L for HeLa and 293T cells, respectively (corresponding IC(50) data of 25 kDa PEI are ca. 11 and 3 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)x(MAc) exhibited higher transfection activity than PEI-(Cys)x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared with 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10% serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient nonviral gene vectors.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

A high throughput microelectroporation device to introduce a chimeric antigen receptor to redirect the specificity of human T cells

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

Cationic methacrylate copolymers containing primary and tertiary amino side groups: Controlled synthesis via RAFT polymerization, DNA condensation, and in vitro gene transfection

AbstractA versatile family of cationic methacrylate copolymers containing varying amounts of primary and tertiary amino side groups were synthesized and investigated for in vitro gene transfection. Two different types of methacrylate copolymers, poly(2‐(dimethylamino)ethyl methacrylate)/aminoethyl methacrylate [P(DMAEMA/AEMA)] and poly(2‐(dimethylamino)ethyl methacrylate)/aminohexyl methacrylate [P(DMAEMA/AHMA)], were obtained by reversible addition‐fragmentation chain transfer (RAFT) copolymerization of dimethylaminoethyl methacrylate (DMAEMA) with N‐(tert‐butoxycarbonyl)aminoethyl methacrylate (Boc‐AEMA) or N‐(tert‐butoxycarbonyl)aminohexyl methacrylate (Boc‐AHMA) followed by acid deprotection. Gel permeation chromatography (GPC) measurements revealed that Boc‐protected methacrylate copolymers had Mn in the range of 16.1–23.0 kDa and low polydispersities of 1.12–1.26. The copolymer compositions were well controlled by monomer feed ratios. Dynamic light scattering and agarose gel electrophoresis measurements demonstrated that these PDMAEMA copolymers had better DNA condensation than PDMAEMA homopolymer. The polyplexes of these copolymers revealed low cytotoxicity at an N/P ratio of 3/1. The in vitro transfection in COS‐7 cells in serum free medium demonstrated significantly enhanced (up to 24‐fold) transfection efficiencies of PDMAEMA copolymer polyplexes as compared with PDMAEMA control. In the presence of 10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) displayed a high transfection activity comparable with or better than 25 kDa PEI. These results suggest that cationic methacrylate copolymers are highly promising for development of safe and efficient nonviral gene transfer agents. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2869–2877, 2010

Differential uptake of DNA–poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces

Genetically modified bone marrow-derived mesenchymal stem cells (MSCs) have proven to be efficient cell carriers for local or systemic delivery of therapeutics as well as growth factors to augment tissue formation. However, efficient non-viral gene transfer to these cells is limiting their applicability. Although most studies have focused on designing more efficient condensation agents for DNA, our focus in this manuscript is to study the role of two extracellular matrix (ECM) proteins, collagen I (Col I) and fibronectin (Fn), on the ability of MSCs to become transfected. Here we report that plating MSCs on Col I-coated surfaces inhibits transfection, while plating MSCs on Fn-coated surfaces enhances transfection. The mechanism by which these ECM proteins affect non-viral gene transfer involves the endocytosis pathway used for polyplex uptake and intracellular tension. We found that Fn promoted internalization through clathrin-mediated endocytosis and that this pathway resulted in more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Further, the disruption of actin-myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn-coated surfaces, but not for cells plated on Col I. We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected and that through understanding the mechanisms by which the ECM affects non-viral gene transfer better materials and transfection protocols can be realized.

Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity

Genetically engineered lymphocytes hold promise for the treatment of genetic disease, viral infections and cancer. However, current methods for genetic transduction of peripheral blood lymphocytes rely on viral vectors, which are hindered by production and safety-related problems. In this study, we demonstrated an efficient novel nonviral platform for gene transfer to lymphocytes. The Sleeping Beauty transposon-mediated approach allowed for long-term stable expression of transgenes at approximately 50% efficiency. Utilizing transposon constructs expressing tumor antigen-specific T-cell receptor genes targeting p53 and MART-1, we demonstrated sustained expression and functional reactivity of transposon-engineered lymphocytes on encountering target antigen presented on tumor cells. We found that transposon- and retroviral-modified lymphocytes had comparable transgene expression and phenotypic function. These results demonstrate the promise of nonviral ex vivo genetic modification of autologous lymphocytes for the treatment of cancer and immunologic disease.

Long-term Transgene Expression in the Central Nervous System Using DNA Nanoparticles

This study demonstrates proof of concept for delivery and expression of compacted plasmid DNA in the central nervous system. Plasmid DNA was compacted with polyethylene glycol substituted lysine 30-mer peptides, forming rod-like nanoparticles with diameters between 8 and 11 nm. Here we show that an intracerebral injection of compacted DNA can transfect both neurons and glia, and can produce transgene expression in the striatum for up to 8 weeks, which was at least 100-fold greater than intracerebral injections of naked DNA plasmids. Bioluminescent imaging (BLI) of injected animals at the 11th postinjection week revealed significantly higher transgene activity in animals receiving compacted DNA plasmids when compared to animals receiving naked DNA. There was minimal evidence of brain inflammation. Intrastriatal injections of a compacted plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) resulted in a significant overexpression of GDNF protein in the striatum 1-3 weeks after injection.

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm(-1) with pre-incubation in Spinner's minimum essential medium buffer at 22 degrees C. Under these conditions beta-galactosidase expression was achieved in 29+/-3% of adherent cells 48 h post transfection. The kinetics of beta-galactosidase activity revealed maintenance of beta-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

Serum-resistant lipopolyplexes for gene delivery to liver tumour cells

In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22 kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22 kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the ζ-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800 kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25 kDa) or linear (22 kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22 kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25 kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.

Efficient Non-Viral Ocular Gene Transfer with Compacted DNA Nanoparticles

BackgroundThe eye is an excellent candidate for gene therapy as it is immune privileged and much of the disease-causing genetics are well understood. Towards this goal, we evaluated the efficiency of compacted DNA nanoparticles as a system for non-viral gene transfer to ocular tissues. The compacted DNA nanoparticles examined here have been shown to be safe and effective in a human clinical trial, have no theoretical limitation on plasmid size, do not provoke immune responses, and can be highly concentrated.Methods and FindingsHere we show that these nanoparticles can be targeted to different tissues within the eye by varying the site of injection. Almost all cell types of the eye were capable of transfection by the nanoparticle and produced robust levels of gene expression that were dose-dependent. Most impressively, subretinal delivery of these nanoparticles transfected nearly all of the photoreceptor population and produced expression levels almost equal to that of rod opsin, the highest expressed gene in the retina.ConclusionsAs no deleterious effects on retinal function were observed, this treatment strategy appears to be clinically viable and provides a highly efficient non-viral technology to safely deliver and express nucleic acids in the retina and other ocular tissues.

Efficient nonviral gene transfer mediated by polyethylenimine in an insect cell line

Efficient nonviral gene transfer mediated by polyethylenimine in an insect cell line

Ultrasound Enhances Gene Delivery of Human Factor IX Plasmid

Delivery of plasmid DNA can be enhanced by treatment with ultrasound (US); acoustic cavitation appears to play an important role in the process. Ultrasound contrast agents (UCAs; stabilized microbubbles) nucleate acoustic cavitation, and lower the acoustic pressure threshold for inertial cavitation occurrence. Fifty micrograms of a liver-specific, high-expressing human factor IX plasmid, pBS-HCRHP-FIXIA, mixed with UCA or phosphate-buffered saline was delivered to mouse livers by intrahepatic injection, with simultaneous exposure to 1 MHz-pulsed US using various acoustic protocols. Variable pulse duration (PD) at constant treatment time, pulse repetition frequency, and an acoustic peak negative pressure amplitude of 1.8 MPa produced 2- to 13-fold enhancements in hFIX gene expression, but PD was not a strong determinant. In contrast, a dose-response relationship was demonstrated for the peak negative pressure (P-), with significant enhancement of gene transduction at P- >/= 2 MPa. Up to 63 ng/ml (approaching the therapeutic range for treating hemophilia patients) could be achieved by transducing one liver lobe at 4-MPa P-, corresponding to a 66- fold increment relative to treatment with naked DNA alone. Under the same conditions, mouse livers could also be transduced with a GFP plasmid. Histology showed transient liver damage caused by intrahepatic injection and US exposure at 4-MPa P-; however, the damage was repaired in a few days. We conclude that therapeutic US in combination with UCA has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia.

Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines

Polyethylenimines (PEIs) are among the most efficient non-viral gene transfer agents developed so far. However, transfections with these polymers were shown to require a very high copy number of plasmid DNA per cell to achieve gene expression. Here, we investigate whether it is possible to reduce the amount of plasmid DNA while keeping a high transfection efficiency. Transfection experiments were performed under various conditions in order to study the interdependence between the amount of reporter DNA, the amine-to-phosphate ratio and the transfection efficiency. When suboptimal amounts of linear PEI 22 kDa/DNA complexes were used for transfection, a severe reduction in reporter gene expression was observed. On the other hand, for optimal amounts of PEI/DNA complexes more than half of the reporter gene can be replaced by carrier DNA or polyglutamic acid without substantially decreasing the transfection efficiency of the polymer both in cultured cells and after systemic administration in mice. When used under the same in vitro experimental conditions, the lipospermine DOGS, but not the monocationic lipid DOTAP, gave similar results. Taken together, our data suggest that the activity of compounds with endosome-buffering capacities, such as PEIs and lipospermines, requires a threshold amount of transfection agent. In addition, our results indicate that, in many gene transfer situations, it will be possible to lower the dose of active plasmid thus reducing costs and the risk of immune stimulation triggered by bacterial DNA.

Efficient Non-Viral Transfection of Mouse and Human Embryonic Stem Cells.

BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells.METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimized on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analyzed by FACS for EGFP expression, viability and phenotype. Also, stably-transfected mouse ES cell lines containing Lox-P or FRT-flanked reporter genes were electroporated with mRNA encoding Cre- or FLPe-recombinase proteins. Monitoring recombination efficiency was done based on the appearance and/or disappearance of fluorescent reporter genes, as determined by FACS analysis. ES cells that underwent recombination were further analyzed for potential to differentiate towards the neural lineage and differentiated cells were analyzed by FACS for expression of neural markers.RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not influence ES cell phenotype and further cell culture of undifferentiated ES cell populations. Electroporation of mRNA encoding Cre- or FLPe-recombinase proteins in stably-transfected mouse ES cell lines containing LoxP- or FRT-flanked reporter genes resulted in a recombination efficiency of respectively 75% and 90%. Moreover, these recombination events did not have influence on ES cell phenotype, viability, growth potential, and their ability to differentiate towards neural cell types upon retinoic acid stimulation. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation.CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cell-fate within in vitro cultured embryoid bodies.