Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.     

Structural basis of importin-α-mediated nuclear transport for Ku70 and Ku80

Ku70 and Ku80 form a heterodimeric complex involved in multiple nuclear processes. This complex plays a key role in DNA repair due to its ability to bind DNA double-strand breaks and facilitate repair by the nonhomologous end-joining pathway. Ku70 and Ku80 have been proposed to contain bipartite and monopartite nuclear localization sequences (NLSs), respectively, that allow them to be translocated to the nucleus independently of each other via the classical importin-α (Impα)/importin-β-mediated nuclear import pathway. To determine the structural basis of the recognition of Ku70 and Ku80 proteins by Impα, we solved the crystal structures of the complexes of Impα with the peptides corresponding to the Ku70 and Ku80 NLSs. Our structural studies confirm the binding of the Ku80 NLS as a classical monopartite NLS but reveal an unexpected binding mode for Ku70 NLS with only one basic cluster bound to the receptor. Both Ku70 and Ku80 therefore contain monopartite NLSs, and sequences outside the basic cluster make favorable interactions with Impα, suggesting that this may be a general feature in monopartite NLSs. We show that the Ku70 NLS has a higher affinity for Impα than the Ku80 NLS, consistent with more extensive interactions in its N-terminal region. The prospect of nuclear import of Ku70 and Ku80 independently of each other provides a powerful regulatory mechanism for the function of the Ku70/Ku80 heterodimer and independent functions of the two proteins.     

An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus

Although the 1235 amino acids human herpes simplex virus type 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism. The present study addresses this issue directly, characterizing pUL30's nuclear import pathway for the first time using quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to a previously described nuclear localization signal (NLS) located within the pUL30 binding site for the polymerase accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (HCMV) DNA polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30-NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip), required for nuclear targeting of pUL30, and able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin (IMP) alpha/beta heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target.     

Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.     

Targeted delivery of plasmid DNA into the nucleus of cells via nuclear localization signal peptide conjugated to DNA intercalating bis- and trisacridines

Efficient nuclear targeting via nonviral delivery of DNA is still an unmet challenge in gene therapy. We have synthesized a novel 9-aminoacridine amino acid monomer that conveniently allows multiple acridines to be incorporated into peptide conjugates. In particular we have prepared bis- and trisacridine conjugates of nuclear localization signal peptide (NLS) ((Acr)2-NLS and (Acr)3-NLS) and studied these as functional transporters for the nuclear delivery of DNA. We show that these conjugates can enhance transfection efficacy as well as nuclear localization of plasmid DNA by more than 50-fold when combined with polyethylenimine at an N:P ratio of 2-3. These conjugates have high reversible affinity for double stranded DNA by intercalation and the technique provides a simple means of associating NLS with DNA of any sequence and at any ratio.     

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.     

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.     

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo

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. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.     

Efficient transfection of primary zebrafish fibroblasts by nucleofection

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.     

Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.     

Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore

One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-β have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Several vectors have been developed in order to target genes to specific cells. Virus-based vectors lead to a high transfection efficiency in vitro, but display important disadvantages such as pathological risks, which they expose to patients. Plasmid-associated chemical vectors lack these disadvantages, but allow only a very low efficiency of transgene expression. Most of the non-viral-based gene transfer techniques developed until now mainly focused their efforts to overcome the problem of DNA entry into the cell. Some recent works, however, have begun to investigate the nucleus entry problem and suggest that the trafficking of DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins. If the vector contains one or several NLS, either as covalently or non-covalently DNA-linked peptides, a competition may take place between the rate of dissociation of the DNA-vector complexes and the rate of loading of the complexes to the NLS-mediated nucleus importation machinery. This equilibrium may be displaced towards the importation pathway by the use of NLS-bearing proteins instead of peptides. The possibility of recruiting normal endogenous cellular pathways of nuclear uptake to promote entry of exogenously applied DNA through the nuclear pore complex would, thus, seem promising. Nevertheless, attempts to improve the transport of DNA to the nucleus through the use of NLSs have achieved limited success. Although these systems show improved transgene expression, little is known about how they function in transfected cells, and the optimal formulation for gene expression is yet to be determined.     

Homodimeric SV40 NLS peptide formed by disulfide bond as enhancer for gene delivery

Recently, cysteine residue incorporation increased liposome-mediated transfection compared to unmodified peptide. Therefore, we designed novel modified SV40 NLS peptides, homodimeric (NLS-CTHD, NLS-NTHD) and closed structure (cyclic NLS), simply using disulfide bond between cysteines to develop more efficient and safe non-viral gene delivery system. The simple mix of NLS-CTHD among these novel transfection enhancing peptides with DNA increased the gene transfer potency of cationic liposomes more efficiently with no additional cytotoxicity.