The FASEB Journal @BULLET Research Communication Nuclear-targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo (original) (raw)
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Faseb Journal, 2008
Poor nuclear entry, especially into nondividing cells, is a limiting factor in nonviral gene delivery. We have engineered a novel chimeric vector relying on the controlled assembly of a TAT-tagged multisubunit DNA binding protein (EcoR124I) with expression plasmids containing the EcoR124I recognition site. Molecular interactions of this molecular assembly were studied by electrophoretic mobility shift assay and atomic force microscopy. Maintenance of nanocomplexes in an appropriate stoichiometric ratio was both necessary and sufficient to produce a significant (>8-fold) increase in the activity of the therapeutic ␣-galactosidase A enzyme after intramuscular administration in the mouse model of Fabry disease. To our knowledge, this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle. Coinjection with pluronic SP1017 produced further enhancement of gene expression, demonstrating cumulative effects of the increased nuclear delivery by TAT chimeras and transcription activation by the pluronic. Cell penetration peptides (CPP), such as TAT, have been shown to improve delivery of macromolecules, when linked directly. However, in our system TAT-enhanced targeting took place even though it was linked to the plasmid DNA molecule indirectly via two noncovalent bonds. Therefore, this proof-of principle result indicates that TAT (and potentially other CPP) can be used for targeting modular chimeric vectors and therapeutic nanodevices.-Lavigne, M. D., Yates, L., Coxhead, P., Gó recki, D. C. Nuclear-targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo. FASEB J. 22,
Nuclear-targeted minicircle to enhance gene transfer with non-viral vectorsin vitro andin vivo
The Journal of Gene Medicine, 2006
Background To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle.
A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
Molecular Therapy, 2011
Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.
DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery
Pharmaceutical Research, 2011
Purpose To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA. Methods A set of DTS was identified and cloned into EGFPreporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry. Results Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNFα, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.
Molecular Pharmaceutics, 2019
Skeletal muscle is ideally suited and highly desirable as a target for therapeutic gene delivery because of its abundance, high vascularization, and high levels of protein expression. However, efficient gene delivery to skeletal muscle remains a current challenge. Besides the major obstacle of cell-specific targeting, efficient intracellular trafficking, or the cytosolic transport of DNA to the nucleus, must be demonstrated. To overcome the challenge of cell-specific targeting, herein we develop a generation 5-polyamidoamine dendrimer (G5-PAMAM) functionalized with a skeletal muscle-targeted peptide, ASSLNIA (G5-SMTP). Specifically, to demonstrate the feasibility of our approach, we prepared a complex of our G5-SMTP dendrimer with a plasmid encoding firefly luciferase and investigated its delivery to skeletal muscle cells. Luciferase assays indicated a threefold increase in transfection efficiency of C2C12 murine skeletal muscle cells using G5-SMTP when compared with nontargeting nanocarriers using unmodified G5. To further improve the transfection yield, we employed a cationic dynein light chain 8 protein (DLC8)-binding peptide (DBP) containing an internal sequence known to bind to the DLC8 of the dynein motor protein complex. Complexation of DBP with our targeting nanocarrier, that is, G5-SMTP, and our luciferase plasmid cargo resulted in a functional nanocarrier that showed an additional sixfold *
Tat-mediated delivery of heterologous proteins into cells
The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to 3-galactosidase, horseradish peroxidase, RNase A, and domain Ill ofPseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-f-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting
Targeted and Stable Gene Delivery into Muscle Cells by a Two-Step Transfer System
Biochemical and Biophysical Research Communications, 2000
We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the -galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of -galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.
Long-term efficient gene delivery using polyethylenimine with modified Tat peptide
Biomaterials, 2014
Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (z5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (z7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.
Recent trends in non‐viral vector‐mediated gene delivery
Biotechnology journal, 2009
Nucleic acids-based next generation biopharmaceuticals (i.e., pDNA, oligonucleotides, short interfering RNA) are potential pioneering materials to cope with various incurable diseases. However, several biological barriers present a challenge for efficient gene delivery. On the other hand, developments in nanotechnology now offer numerous non-viral vectors that have been fabricated and found capable of transmitting the biopharmaceuticals into the cell and even into specific subcellular compartments like mitochondria. This overview illustrates cellular barriers and current status of non-viral gene vectors, i.e., lipoplexes, liposomes, polyplexes, and nanoparticles, to relocate therapeutic DNA-based nanomedicine into the target cell. Despite the awesome impact of physical methods (i.e., ultrasound, electroporation), chemical methods have been shown to accomplish high-level and safe transgene expression. Further comprehension of barriers and the mechanism of cellular uptake will facilitate development of nucleic acids-based nanotherapy for alleviation of various disorders.