Winfried Wünsche

Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.

Increased RNAi is related to intracellular release of siRNA via a covalently attached signal peptide

In the last decade short interfering RNA (siRNA) became an important means for functional genomics and the development of gene-specific drugs. However, major technical hurdles in the application of siRNA include its cellular delivery followed by its intracellular trafficking and its release in order to enter the RNA interference (RNAi) machinery. The novel phosphorothioate-stimulated cellular uptake of siRNA contrasts other known delivery systems because it involves a caveosomal pathway in which large amounts of siRNA are delivered to the perinuclear environment, leading to measurable though moderate target suppression. Limited efficacy seems to be related to intracellular trapping of siRNA. To study the role of intracellular trafficking of siRNA for biological effectiveness we studied whether a signal peptide for trans-membrane transport of bacterial protein toxins, which is covalently attached to siRNA, can promote its release from the perinuclear space into the cytoplasm and thereby enhance its biological effectiveness. We show that attachment of the peptide TQIENLKEKG to lamin A/C-directed siRNA improves target inhibition after its PS-stimulated delivery. This is related to increased efflux of the siRNA-peptide conjugate from the ER-specific perinuclear sites. In summary, this study strongly suggests that intracellular release of siRNA leads to increased biological effectiveness. Thus covalent peptide-siRNA conjugates are proposed as new tools to study the relationship between intracellular transport and efficacy of siRNA.

Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO2), cycloheximide (CHX) and LipofectamineTM 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi. Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO2. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

Concentrations of circulating RNA from healthy donors and cancer patients estimated by different methods.

Abstract:  Circulating RNA (cirRNA) was isolated from plasma and cell surface‐bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki‐67 mRNA, and 18S rRNA were measured by real‐time quantitative PCR (RT‐qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki‐67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki‐67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki‐67 mRNA correlated with the concentration of total RNA only in the cell surface‐bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface‐bound fraction and blood. The copy number of Ki‐67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki‐67 mRNA and 18S rRNA was found only in cell surface‐bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT‐qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer‐specific mRNA among patients.

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3Cpro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G5T) as a 3Cpro protease inhibitor. Using 1H NMR spectroscopy, G5T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of 1H, 15N-HSQC experiments the binding site for G5T was located to the C-terminal β-barrel of HAV 3Cpro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G5T-binding site, nor does G5T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.

Specific recognition of proteins by array-bound hexanucleotides.

Maximum information density: The complete hexanucleotide sequence space can be immobilized on a chip. The binding of a number of proteins was profiled with this array of 4096 sequences, including fluorescently labeled forms of the HIV‐2 and HIV‐1 reverse transciptases (the consensus motif is shown in the picture). This new technique should aid in the search for pharmaceutical lead compunds.WILEY-VCH

A hexanucleotide selected for increased cellular uptake in cis contains a highly active CpG-motif in human B cells and primary peripheral blood mononuclear cells

The relationship between immunostimulation of human B cells by cytosine–phosphate–guanosine (CpG) ‐containing oligonucleotides and their physical cellular uptake is of mechanistic interest and a prerequisite for rational improvements of the therapeutic potential of CpG‐harbouring oligonucleotides. Here, a combinatorial approach was used to identify nucleotide sequence motifs that facilitate increased cellular uptake in mammalian cells. Oligonucleotides harbouring the selected hexanucleotide TCGTGT in cis show increased cellular uptake. This motif contains a CpG dinucleotide within a sequence context that shows a very strong CpG‐specific stimulatory activity on human B cells. Here we describe the influence of concentration, length and sequence position of the unmethylated CpG dinucleotide on immunostimulation. A comparison between phosphorothioate‐derivatives and unmodified TCGTGT‐containing oligonucleotides strongly indicates a great CpG‐specificity for the unmodified CpG‐harbouring oligonucleotides but not for the phosphorothioate versions. This work describes a link between the physical cellular uptake of naked oligonucleotides harbouring the selected cellular uptake motif TCGTGT, its strong CpG‐specific stimulation of human B cells and its relationship with the sequence context of CpG and its cellular uptake.