Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R(6)-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition

A clearing house for diagnostic testing: the solution to ensure access to and use of patented genetic inventions?

In genetic diagnostics, the emergence of a so-called "patent thicket" is imminent. Such an overlapping set of patent rights may have restrictive effects on further research and development of diagnostic tests, and the provision of clinical diagnostic services. Currently, two models that may facilitate access to and use of patented genetic inventions are attracting much debate in various national and international fora: patent pools and clearing houses. In this article, we explore the concept of clearing houses. Several types of clearing houses are identified. First, we describe and discuss two types that would provide access to information on the patented inventions: the information clearing house and the technology exchange clearing house. Second, three types of clearing houses are analysed that not only offer access to information but also provide an instrument to facilitate the use of the patented inventions: the open access clearing house, the standardized licences clearing house and the royalty collection clearing house. A royalty collection clearing house for genetic diagnostic testing would be the most comprehensive as it would serve several functions: identifying patents and patent claims essential to diagnostic testing, matching licensees with licensors, developing and supplying standardized licences, collecting royalties, monitoring whether users respect licensing conditions, and providing dispute resolution services such as mediation and arbitration. In this way, it might function as an effective model for users to facilitate access to and use of the patented inventions. However, it remains to be seen whether patent holders with a strong patent portfolio will be convinced by the advantages of the royalty collection clearing house and be willing to participate

Dealing with patent fragmentation in ICT and genetics: Patent pools and clearing houses

Recent years have shown a remarkable increase of patents in the field of ICT and genetics. The omnipresence of patents in those areas has raised serious concerns about access to and use of ICT and genome related inventions, as the expansion of patents in those fields might result in a patent thicket. Collaborative licensing models, such as patent pools and clearing houses, have attracted great attention, as they might serve as a mechanism to deal with patent thickets in ICT and genetics and facilitate access to ICT and genomic patents. It remains to be seen to what extent the lessons from the collaborative rights experiments in ICT and genetics, can be applied in a cyberinfrastructure setting

A clearing house for diagnostic testing: the solution to ensure access to and use of patented genetic inventions?

In genetic diagnostics, the emergence of a so-called "patent thicket" is imminent. Such an overlapping set of patent rights may have restrictive effects on further research and development of diagnostic tests, and the provision of clinical diagnostic services. Currently, two models that may facilitate access to and use of patented genetic inventions are attracting much debate in various national and international fora: patent pools and clearing houses. In this article, we explore the concept of clearing houses. Several types of clearing houses are identified. First, we describe and discuss two types that would provide access to information on the patented inventions: the information clearing house and the technology exchange clearing house. Second, three types of clearing houses are analysed that not only offer access to information but also provide an instrument to facilitate the use of the patented inventions: the open access clearing house, the standardized licences clearing house and the royalty collection clearing house. A royalty collection clearing house for genetic diagnostic testing would be the most comprehensive as it would serve several functions: identifying patents and patent claims essential to diagnostic testing, matching licensees with licensors, developing and supplying standardized licences, collecting royalties, monitoring whether users respect licensing conditions, and providing dispute resolution services such as mediation and arbitration. In this way, it might function as an effective model for users to facilitate access to and use of the patented inventions. However, it remains to be seen whether patent holders with a strong patent portfolio will be convinced by the advantages of the royalty collection clearing house and be willing to participate

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.Laboratorium voor Medicinale chemie.status: publishe

-activation inhibitory effects of disulfide-linked CPP–PNA conjugates – in the HeLa cell reporter assay with 6 h delivery () or 24 h ()

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Firefly luciferase activity represents Tat-TAR dependent expression whilst luciferase activity represents control constitutive expression. Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM CPP–PNA concentrations

Confocal microscopy images of the uptake of fluorescein (FAM)-labelled CPP–PNA conjugates when incubated for 5

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>5 h with unfixed HeLa cells. Nuclei are stained red with hydroethidine. ( and ) Orange filter to view both the red colour of hydroethidine and the fluorescein fluorescence. ( and ) Green filter to view only the fluorescein fluorescence. (A and B) Show incubations in the absence of chloroquine, (C and D) Show incubations in the presence of 100 µM chloroquine. First line Tat–PNA (); second line Transportan–PNA (); third line NLS–PNA–Tat (); fourth line K–PNA (). In (C), second line, yellow dots are marked with arrows showing co-localization of hydroethidine dye and fluorescein fluorescence on the inner wall of the nucleus in several nuclei