Nonviral Gene Transfer Vectors Research Articles

Gene Therapy for Intervertebral Disc Degeneration: An Overview of Current Strategies and Applications

Intervertebral disc degeneration is a pervasive condition contributing to chronic back pain, affecting up to a third of the population, with risk further increasing with age. It is a significant driver of disability for millions of Americans and others worldwide. The standard of care today is reliant on symptomatic treatment rather than addressing the root cause of disease. Surgical interventions alter the structural integrity and biomechanics of the spine, often leading to loss of function and motion, and post-operative complications. This is the basis for innovation in novel biologic treatments, including gene therapy, which aims to reestablish the optimal balance between matrix catabolism and anabolism within pathologically degenerating disc cells. This review will cover the significant advances that have led to identification of target therapeutic genes combined with regulated expression of the therapeutic transgene and successful systems for gene delivery into cells. Recent advances in viral and non-viral vectors for gene transfer, silencing of genes by RNA interference, editing of genes by clustered regularly interspaced short palindromic repeats, and modifying mammalian target of rapamycin signaling pathwayS offer promising treatment avenues. Clinical translation of these approaches, however, will require further investigation of the pathological basis of disc degeneration in addition to systematic safety measures for the adoption of gene therapy.

Research advance in lipid nanoparticle-mRNA delivery system and its application in CAR-T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has shown significant efficacy for hematological malignancies, however, it needs to be further optimized. Recently, the lipid nanoparticle (LNP)-mRNA delivery system as a nonviral gene transfer vector has gained rapid progress in CAR-T cell therapy. The claudin-6 (CLDN6) mRNA is delivered to antigen presenting cells (APCs) through LNP system, thereby enhancing the function of CLDN6 CAR-T cells for the clearance of solid tumor cells. For treatment of acute cardiac injury, the fibroblast activation protein (FAP) CAR mRNA can be delivered to T cells through LNP system for the in vivo production of FAP CAR-T cells, thereby blocking the process of myocardial fibrosis. The LNP-mRNA delivery system has advantages including having no integration in host genome, inexpensiveness, low toxicity and modifiability; on the other hand, it has certain disadvantages such as limited cell persistence caused by transient protein expression and limitations in preparation techniques. This article reviews the research advance in LNP-mRNA in vivo delivery system and its application in CAR-T cell therapy.

Soft-Matter Nanotubes: A Platform for Diverse Functions and Applications

Self-assembled organic nanotubes made of single or multiple molecular components can be classified into soft-matter nanotubes (SMNTs) by contrast with hard-matter nanotubes, such as carbon and other inorganic nanotubes. To date, diverse self-assembly processes and elaborate template procedures using rationally designed organic molecules have produced suitable tubular architectures with definite dimensions, structural complexity, and hierarchy for expected functions and applications. Herein, we comprehensively discuss every functions and possible applications of a wide range of SMNTs as bulk materials or single components. This Review highlights valuable contributions mainly in the past decade. Fifteen different families of SMNTs are discussed from the viewpoints of chemical, physical, biological, and medical applications, as well as action fields (e.g., interior, wall, exterior, whole structure, and ensemble of nanotubes). Chemical applications of the SMNTs are associated with encapsulating materials and sensors. SMNTs also behave, while sometimes undergoing morphological transformation, as a catalyst, template, liquid crystal, hydro-/organogel, superhydrophobic surface, and micron size engine. Physical functions pertain to ferro-/piezoelectricity and energy migration/storage, leading to the applications to electrodes or supercapacitors, and mechanical reinforcement. Biological functions involve artificial chaperone, transmembrane transport, nanochannels, and channel reactors. Finally, medical functions range over drug delivery, nonviral gene transfer vector, and virus trap.

Gene Therapy 2017: Progress and Future Directions.

Gene therapy has changed dramatically in the 28 years since the first human gene transfer experiment in 1989. Alipogene tiparvovec, GlyberaR®, a recombinant adeno‐associated virus (rAAV) product for lipoprotein lipase deficiency, and Strimvelis®, a lentivirus vector for severe combined immune deficiency are approved in Europe. An rAAV2 product for a congenital form of blindness is currently under review in the United States, likely to be followed by numerous other gene therapies. Nonviral gene transfer The success of gene therapy has largely been driven by improvements in nonviral and viral gene transfer vectors. An array of physical and chemical nonviral methods have been used to transfer DNA and mRNA to mammalian cells and a substantial number of these have been developed as clinical stage technologies for gene therapy, both ex vivo and in vivo. Cationic liposome technology is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co‐lipid, thought to enhance liposome uptake by mammalian cells.4, 5, 6, 7 Similarly, other polycations, such as poly‐l‐lysine and polyethylene‐imine, complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome‐mediated uptake.8 Several of these cationic‐nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA.9, 10, 11 Modified (and unmodified or “naked”) DNA, RNA, and oligonucleotides have also been shown to mediate successful gene transfer in a number of circumstances. These include the use of pDNA by direct intramuscular injection for DNA vaccines, the use of intratumoral injection of pDNA to deliver cytokine and/or suicide genes, systemic (s.c. or i.v.) injection of antisense nucleotides to induce RNAse H1 or exon‐skipping.12, 13, 14 The most recent of these developed for induction of RNAi are discussed in a later section. Ex vivo introduction of pDNA and/or other nucleotides using physical methods has been well developed for certain cell types, including T lymphocytes.15 Electroporation techniques have become the standard with T cells for the introduction of a variety of molecular cargoes, including ribonucleoproteins composed of Cas9 and short‐guide RNAs for genome editing (see section below) and transposons for long‐term integration of transgenes.

Graphene materials as 2D non-viral gene transfer vector platforms.

Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.

Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

Nanoparticle siRNA against BMI-1 with a Polyethylenimine-Laminarin Conjugate for Gene Therapy in Human Breast Cancer.

The B-cell-specific Moloney leukemia virus inset site 1 gene (BMI-1) has attracted considerable attention in recent years because of its key role in breast cancer development and metastasis. The downregulation of BMI-1 expression via small interfering RNA (siRNA) effectively inhibits tumor growth. However, the successful application of this therapy is limited by the unavailability of an appropriate vector for siRNA transfer. Therefore, this study aimed to construct a novel laminarin-based nonviral gene transfer vector to carry a constructed BMI-1-targeting siRNA and to investigate the in vitro and in vivo antitumor effects of this siRNA on breast cancer cells. To enhance the siRNA-carrying capacity, we introduced polyethylenimine (PEI) to laminarin's surface via N,N'-carbonyldiimidazole, which produced the cationic PEI-modified laminarin conjugate nLP. Subsequent in vitro experiments indicated that nLP not only formed a nanoparticle with a diameter of 200 nm through electrostatic interactions with siRNA but also showed high efficiency (95.0%) in the delivery siRNA to MCF-7 cells. The nanoparticle targeting BMI-1 (nLP/siBMI-2) reduced BMI-1 expression in breast MCF-7 cells by 90.9% reduction. An in vivo tumor suppression experiment demonstrated that the nLP/siBMI-2 nanoparticle had relatively low toxicity and good gene-therapeutic efficacy, with a tumor inhibition rate of 46.6%.

Gene-Based Therapy for Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin Deficiency (AATD) has been an attractive target for the development of gene therapy because it is a common single gene disorder, for which there would appear to be significant benefit to be gained for lung disease patients by augmentation of plasma levels of wild-type (M) alpha-1 antitrypsin (AAT). While a significant proportion of patients also have liver disease, which is unlikely to be benefitted by augmentation, the potential to treat or prevent lung disease by replacement of plasma levels to at least 11 microMolar (571mcg/ml) is the basis upon which several protein replacement therapies have been licensed for human use. Further enhancing the likelihood of success of gene therapy is the fact that the AAT coding sequence is relatively short and the protein appears to function primarily in the plasma and extracellular space. This means that AAT production from any cell or tissue capable of secreting it could be useful therapeutically for augmentation. Based on these considerations, attempts have been made to develop AAT therapies using nonviral gene transfer, gammaretrovirus, recombinant adenovirus (rAd), and recombinant adeno-associated virus (rAAV) vectors. These have resulted in three phase I clinical trials (one of cationic liposome, one of rAAV2, and one of rAAV1) and one phase II clinical trial (with rAAV1). The results of the latter trial, while promising, demonstrated levels were only 3 to 5% of the target range. This indicates the need to further increase the dose of the vector and/or to increase the levels to within the therapeutic range.

Tell Me Something I Do Not Know. Multiscale Molecular Modeling of Dendrimer/ Dendron Organization and Self-Assembly In Gene Therapy

Due to the relative easy synthesis and commercial availability, nanovectors based on dendrimers and dendrons are among the most utilized non-viral vectors for gene transfer. Contextually, recent advances in molecular simulations and computer architectures not only allow for accurate predictions of many structural, energetical, and eventual self-assembly features of these nanocarriers per se, but are able to yield vital (and perhaps otherwise unattainable) molecular information about the interactions of these nanovectors with their nucleic acid cargoes. In the present work, we aim at reviewing our own efforts in the field of multiscale molecular modeling of these interesting materials. In particular, our originally developed computational recipes will be presented, and the link between simulations and experiments will be described and discussed in detail. This review is written by computational scientists for experimental scientists, with the specific purpose of illustrating the potentiality of these methodologies and the usefulness of multiscale molecular modeling as an innovative and complementary tool in their current research.

Nonviral gene delivery using poly-D /L aspartate-diethylenetriamine cationic polymers and polyethylene glycol: A two-step approach

Cationic polymers have received much attention as promising nonviral vectors for gene transfer. However, development of polymers with low cell toxicities and high transfection efficiencies continue to be a significant problem and a major hurdle to their success. Poly-D/L aspartate-diethylenetriamine poly(D/L Asp-DET) polymers were synthesized and evaluated as nonviral gene delivery agents. Poly(D/L Asp-DET) polymers display endosome buffering capacity. The polymers condense plasmid DNA above N:P ratios of 1 and form polyplex particles of ∼50–100 nm, with zeta potentials between neutral and +40 mV. Transmission electron microscopy shows the polyplexes to be uniform in size and shape. Polyplexes maintain the structural integrity of DNA following incubation in nucleases and also show high transfection efficiencies with minimal toxicity in both HCT-116 and PC-3 cell culture. However, it is found that these poly(D/L Asp-DET)/DNA polyplexes immediately aggregate in salt and serum conditions, making them unsuitable for use in vivo. Therefore, the polyplexes were further modified by covalent addition of polyethylene glycol (PEG). Introduction of this second step produces PEG-polyplexes of uniform size (below 100 nm), with neutral zeta potentials that are also stable in both salt and serum conditions. These results suggest poly(D/L Asp-DET) cationic polymers as potentially safe and efficient nonviral gene delivery agents. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

Functionalized organic nanotubes as tubular nonviral gene transfer vector

Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1 μm in length was rarely taken up into the cells, while those with a length of 400–800 nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.

Abstract 5408: PEG surface modification of Poly(D/L Asp-DET)/DNA polyplexes greatly increases tumor accumulation and improves biodistribution

Abstract Cationic polymers have received much attention as promising non-viral vectors for gene transfer. However, generating polymers that are stable and can systemically deliver large amounts of nucleic acids to specific disease sites continues to be a significant hurdle to non-viral vector success. Therefore, the purpose of this study was to develop a new biocompatible and biodegradable cationic polymer system for non-viral gene delivery of plasmid DNA. Poly-d/l Aspartate-Diethylenetriamine [Poly(d/l Asp-DET)] polymers were synthesized and used to complex plasmid DNA. Polymer/DNA polyplexes were further modified by covalent coupling with various polyethylene glycol (PEG) polymers. Polyplexes were characterized and evaluated for particle size, zeta potential and morphology by TEM. Plasmid DNA stability was assessed by challenging polyplexes with serum nucleases, and the efficacy of DNA delivery and gene expression was examined by performing transfection experiments on cancer cells. In vivo DNA tumor delivery and biodistribution was also investigated using tumor bearing nude mice. Poly(d/l Asp-DET) polymers were able to bind to plasmid DNA and formed polyplex particles the were amenable to surface coating with PEG. PEG-Polyplexes were approximately ∼70nm in diameter with neutral zeta potentials. TEM confirmed that the polyplexes were uniform in size. PEG-Polyplexes were also extremely stable and maintained the structural integrity of DNA following incubation in nucleases, and were also capable of transfecting HCT-116 and PC-3 culture cells with no cell toxicity. Tail vein administration of PEG-Polyplexes to tumor bearing nude mice did not reveal any observable toxicities. Moreover, improvements in PEG polymer chemistry increased nucleic acid accumulation in tumors while reducing DNA accumulation in other tissues including liver, kidney and spleen. Poly(d/l Asp-DET) cationic polymers show enormous potential as non-viral drug delivery agents. Further, the surface modification of polyplexes with PEG allowed for sustained systemic delivery of nucleic acids to tumors. The characterization of PEG-Poly(Asp-DET)/DNA polyplexes provides useful insights that can be used to the design vectors for targeted nucleic acid delivery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2011-5408

Nucleolin-Mediated Cellular Trafficking of DNA Nanoparticle Is Lipid Raft and Microtubule Dependent and Can Be Modulated by Glucocorticoid

DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin-DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery.

Abstract 599: Development of poly(Asp-DET) cationic polymers for nonviral gene delivery

Abstract Cationic polymers have received much attention as promising non-viral vectors for gene transfer. However, identification of polymers able to systemically deliver nucleic acids continues to be a significant hurdle to the success of this methodology. The purpose of this study was to develop a new biocompatible and biodegradable cationic polymer system as a non-viral gene delivery agent for plasmid DNA. Poly-aspartate-diethylene triamine (“P(Asp-DET)”) polymers were synthesized and utilized to complex plasmid DNA. The resulting polymer/DNA complexes, also known as “polyplexes,” were further modified by covalent coupling with polyethylene glycol (PEG), known as “PEGylation,” and characterized. Polyplexes were evaluated for efficiency of encapsulation, particle size, zeta potential and morphology by TEM. Stability of the complexed plasmid DNA was assessed by challenging polyplexes with nucleases. Efficacy of DNA delivery and gene expression was examined by performing transfection experiments using HCT-116 cells. In vivo DNA delivery was also investigated using tumor-bearing nude mice. Asp-DET polymers were found to bind to plasmid DNA with polymer to DNA (+/-) charge ratios of above 1.5. In addition, these polymers formed polyplex particles of approximately 50-150 nm with zeta potentials between neutral and +40 mV. TEM showed that the polyplexes were uniform and spherical in shape. It was also demonstrated that the polyplexes maintained the structural integrity of DNA following incubation in nucleases. The polyplexes were also capable of transfecting HCT-116 culture cells, with non-PEG complexes having significantly higher GFP and luciferase expression. Tail vein injections of post-PEGylated polyplexes into tumor bearing nude mice did not reveal any observable toxicities. Notably, nucleic acid accumulation was found in tumors with expression detected in lymph nodes. P(Asp-DET) cationic polymers show enormous potential as non-viral drug delivery agents. Further, the surface modification of described polyplexes with PEG allowed for systemic delivery of nucleic acids to tumors. The characterization of post-PEGylated Asp-DET/DNA polyplexes provides insights that can be used to design vectors for targeted nucleic acid delivery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 599.

PiggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery

To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity. Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry. The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors. L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.

Comparison of Viral and Nonviral Vectors for Gene Transfer to Human Endothelial Progenitor Cells

The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery. In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro. We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.

Comparison between Cationic Polymer and Lipid in Plasmidic DNA Delivery to the Cell Nucleus

We investigated the cell association and intracellular pathway to the nucleus of complexes formed between DNA and cationic lipid DOTAP (lipoplexes) or cationic polymer polyethylenimine (polyplexes). Flow cytometry and confocal microscope analysis showed that lipoplexes presented higher affinity for cell membrane than polyplexes. Electron microscopy demonstrated that both types of complexes followed an endocytic pathway and were metabolized but did not enter the nucleus. However, by in situ PCR and FISH it was possible to show that the plasmid localized to the nuclei, indicating that DNA must be dissociated from the vectors to be delivered to the nucleus. Our results identify a different behaviour in the interaction of polyplexes and lipoplexes with the cell, indicating that a combination of advantageous properties of the two kinds of cationic molecules could further ameliorate efficiency of nonviral gene transfer vectors.

RNA Interference Screen to Identify Pathways That Enhance or Reduce Nonviral Gene Transfer During Lipofection

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle gamma-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.