A Biological Transporter for the Delivery of Peptide Nucleic Acids (PNAs) to the Nuclear Compartment of Living Cells

Braun, Klaus; Peschke, Peter; Pipkorn, Rüdiger; Lampel, Stefan; Wachsmuth, Malte; Waldeck, Waldemar; Friedrich, E.; Debus, Jürgen

doi:10.1016/s0022-2836(02)00031-1

Cited by 90 publications

(78 citation statements)

References 33 publications

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“…The use of a low MOI allowed us to study viral dissemination occurring after primary infection. On days 1, 3 (before and after repeated changes of the culture medium), 4,6,8,10,12, and 14, samples were collected (Fig. 1A) and analyzed by immunofluorescence microscopy, and virus spread was quantified.…”

Section: Resultsmentioning

confidence: 99%

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

Bajramović¹,

Münter

Syan³

et al. 2003

J Virol

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Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.Borna disease virus (BDV) is the etiological agent of Borna disease, a neurological disorder of horses, sheep, and other farm animals (27). Recent evidence indicates that the natural host range, the geographic distribution, and the prevalence of BDV are much broader than previously thought (21, 40, 45). The spectrum of clinical diseases due to BDV infection ranges from subtle behavioral abnormalities (e.g., impairment of learning and memory) to progressive, immune-mediated meningoencephalitis (17, 38). There is considerable evidence that BDV also infects humans (5, 9, 36). Although it has been linked to certain neuropsychiatric disorders, the epidemiology and the clinical consequences of human infection with BDV remain controversial (2,3,5,26,41).BDV is a nonsegmented negative-strand RNA virus (8, 43) that has the property, unique among members of the order Mononegavirales that infect animals, of transcribing and replicating its genome in the nucleus (7). The BDV genome consists of an 8.9-kb RNA, which includes at least six open reading frames (ORFs) (23). ORF I codes for the nucleoprotein (N) p40, ORF II codes for the phosphoprotein (P) p24, ORF III codes for the matrix protein p16, ORF IV codes for the glycoprotein (GP) p56, ORF V codes for the polymerase protein p180/190, and an ORF overlapping with the P gene codes for protein p10. Characterization of amino acid sequences of several BDV strains and isolates from various parts of the world revealed that BDV is a remarkably conserved virus with 84 to 95% amino acid identities among all gene pr...

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Section: Resultsmentioning

confidence: 99%

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

Bajramović¹,

Münter

Syan³

et al. 2003

J Virol

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“…[13][14][15] In order to improve delivery of PNAs into cells and to reduce the doses required for detectable effects, several chemical modifications based on covalently linked cell-penetrating peptides have been successfully used. 10,12,[16][17][18] Linkage of nuclear localization or polylysine sequences to PNAs have also been reported to improve both their cellular internalization and nuclear translocation. 14,19,20 Moreover, it has recently been shown that the cellular uptake of PNAs depends on the cell type and that a carrier system is required for efficient uptake.…”

Section: Introductionmentioning

confidence: 99%

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

et al. 2004

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The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNApPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNApPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipidbased methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.

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“…For the most part, PNAs have been used as an antigene agent (14-17), because they have the capacity for down-regulating gene expression in cultured cells and in animals. In addition, PNAs have been modified to maximize cellular/nuclear entry in order to increase the efficiency for in vivo applications (15,18,19). However, currently there is no established PNA conjugated system that combines these varied modifications and has been demonstrated to stably and efficiently enter, target, and transcriptionally activate an endogenous chromosomal locus in living cells.…”

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confidence: 99%

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Chen

Peterson²,

Iancu‐Rubin³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNAbinding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.γ-globin gene reactivation | primary human CD34+ cells

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A Biological Transporter for the Delivery of Peptide Nucleic Acids (PNAs) to the Nuclear Compartment of Living Cells

Cited by 90 publications

References 33 publications

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

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