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1.
Curr Pharm Des ; 11(28): 3671-80, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16305503

RESUMO

Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.


Assuntos
DNA/administração & dosagem , DNA/química , Terapia Genética , Peptídeos/síntese química , Peptídeos/farmacologia , Animais , Assialoglicoproteínas/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnicas de Química Combinatória , Sistemas de Liberação de Medicamentos , Humanos , Fígado/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/farmacologia
2.
Biomol Eng ; 22(5-6): 185-92, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16144773

RESUMO

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.


Assuntos
DNA Super-Helicoidal/química , Terapia Genética , Ácidos Nucleicos Peptídicos/química , Plasmídeos/química , Animais , Humanos , Cinética , Hibridização de Ácido Nucleico
3.
Glycoconj J ; 21(5): 227-41, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15486455

RESUMO

In order to develop the non-viral Bioplex vector system for targeted delivery of genes to hepatocytes, we have evaluated the structure-function relationship for a number of synthetic ligands designed for specific interaction with the hepatic lectin ASGPr. Biotinylated ligand derivatives containing two, three or six beta-linked N-acetylgalactosamine (GalNAc) residues were synthesized, bound to fluorescent-labeled streptavidin and tested for binding and uptake to HepG2 cells using flow cytometry analysis (FACS). Uptake efficiency increased with number of displayed GalNAc units per ligand, in a receptor dependent manner. Thus, a derivative displaying six GalNAc units showed the highest uptake efficacy both in terms of number of internalizing cells and increased amount of material taken up per each cell. However, this higher efficiency was shown to be due not so much to higher number of sugar units, but to higher accessibility of the sugar units for interaction with the receptor (longer spacer). Improving the flexibility and accessibility of a trimeric GalNAc ligand through use of a longer spacer markedly influenced the uptake efficiency, while increasing the number of GalNAc units per ligand above three only provided a minor contribution to the overall affinity. We hereby report the details of the chemical synthesis of the ligands and the structure-function studies in vitro.


Assuntos
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/metabolismo , Marcação de Genes , Glicosídeos/síntese química , Acetilgalactosamina/metabolismo , Receptor de Asialoglicoproteína/química , Biotinilação , Linhagem Celular Tumoral , Células Cultivadas , Glicosídeos/química , Glicosídeos/metabolismo , Células HeLa , Humanos , Ligantes , Ácidos Nucleicos Peptídicos/química
4.
Biomol Eng ; 21(2): 51-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15113558

RESUMO

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.


Assuntos
DNA Super-Helicoidal/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Plasmídeos/química , Tiazóis/química , Benzotiazóis , Técnicas de Transferência de Genes , Cinética , Hibridização de Ácido Nucleico , Quinolinas
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