Alfaviirustel põhinevate multifunktsionaalsete geeniekspressiooni ja -teraapia süsteemide väljatöötamine ja funktsionaalne analüüs

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Alfaviirused on positiivse polaarsusega RNA genoomiga viirused sugukonnast Togaviridea. Looduses levivad alfaviirused lülijalgsete vektorite abil. Selgroogsetel peremeestel põhjustavad nad erinevaid haigusi nagu entsefaliit ja artriit. Tänu oma headele omadustele on alfaviirused kujunenud perspektiivseteks tööriistadeks geeni- ja kasvajavastaste vaktsiinide väljatöötamisel. Uurimus töös kajastati SFV genoomi cDNA-l põhineva infektsioonilise DNA/RNA kihtvektori ning selle alusel valmistatud rekombinantsete vektorite kavandamist, valmistamist ja iseloomustamist. Esmaseks eesmärgiks oli välja selgitada põhjused, mis muudavad pSP6-SFV4 bakterirakkudes geneetiliselt ebastabiilseks. Tehti kindlaks, et seda põhjustab SFV membraanivalkude soovimatust ekspressioonist tulenev toksiline mõju bakterile. See probleem lahendati liites SFV lugemisraami introni, mis takistas toksiliste valkude tootmist bakteris kuid ei mõjutanud negatiivselt loodud DNA/RNA kihtvektorist vabanenud viiruse omadusi. Loodud töövahendit kasutati järgnevates uurimustöödes, mille ühiseks eesmärgiks oli vektorite ohutuse tagamine. Selleks kavandati ja konstrueeriti SFV põhised DNA/RNA kihtvektorid milles rekombinantse SFV genoomi vabanemist takistavad viiruse kodeerivasse alasse sisse viidud defektsed intronid, mis suruvad efektiivselt maha viiruse infektsioonilise RNA vabanemist ja splaisingu mustrit muutvate oligonukleotiidide lisamine vähendab oluliselt või kõrvaldab selliste intronite põhjustatud negatiivse mõju viirus-vektorile. Raku micro-RNAde kasutamine DNA/RNA kihtvektorites võimaldab viiruse paljunemist kontrollida ka peale tema infektsioonilise genoomi vabanemist, mis on saavutatav liites viiruse genoomi raku miRNAde sihtmärk-järjestusi.Alphaviruses are small positive-strand RNA viruses that make up the genus Alphavirus of the family Togaviridae. Alphaviruses infect a large variety of cell types within organisms. This property makes it possible to use these viruses to develop replication-competent vectors that may have clinical applications. This thesis is based on three studies dedicated to the development and analysis of infectious DNA/RNA layered clone of SFV4 and a panel of vector constructs based on this clone. The first aim was identifying and subsequently eliminating the reasons for the instability of pSP6-SFV4 in bacterial cells. It was found that the instability of this plasmid was caused by the toxic effects of SFV envelope proteins that were cryptically expressed in bacterial cells. The problem was eliminated by interrupting the corresponding reading frame with an intron; this manipulation proved to have no adverse effect on the SFV4 rescued from the obtained DNA/RNA layered vector. A central problem hampering the medical use of alphavirus vectors is concern over their safety issues associated with the fact that alphavirus-based vectors are very difficult to regulate and control. Therefore we were designed, constructed and tested DNA/RNA layered vectors, in which the rescue of replication-competent RNA genome of SFV was efficiently inhibited by aberrantly spliced introns and the delivery of splice-switch oligonucleotides completely or almost completely restored the infectivity of the vector. Using cellular miRNAs to target the genomes of recombinant alphavirus may also represent a viable method to control rescue as well as replication and spread of the vector. It was found that repression of virus replication can be achieved by the insertion of different types of miRNA targets, including those naturally occurring in cellular mRNAs

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50) : 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50 : 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide: RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.Peer reviewe

Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability

Illustration of used miR target cassette design.

<p>Identical miRNA target sites are shown using the same colour code (grey, white or striped); the 8-nucleotide spacers between miRNA target sites are shown in black.</p

Northern blot analysis of SFV RNAs in transfected cells.

<p>BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.</p

Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets.

<p>(A) General design of the vectors; the same symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075802#pone-0075802-g003" target="_blank">Fig. 3A</a> are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and HeLa (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p<0.05), as determined by a Mann – Whitney U test.</p

Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors.

<p>BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to <i>Let-7</i>, <i>miR-17</i> and <i>miR-19</i> (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.</p

Insertion of miR-1cl and miR-cl2 cassettes suppresses Gluc expression from a non-viral vector.

<p>(A) Schematic representation of non-viral expression vector (above) and corresponding mRNA (below). The plasmid backbone of the vector is not shown. CMV, immediate-early promoter of human cytomegalovirus; Rz, negative strand ribozyme of hepatitis delta virus (cleavage site is indicated with bent arrow); p(A), early polyadenylation signal of simian virus 40; (A)n, poly(A) tail. The 5′ and 3′ UTRs of the transcribed mRNA correspond to those of SFV4. Effects of miR-cl1 target sequences on Gluc expression in transfected BHK-21 (B) or HeLa cells (C) and effects of miR-cl2 target sequences on Gluc expression in transfected BHK-21 (D) or HeLa cells (E) were analysed as follows. Cells were transfected by electroporation, and Gluc activity in the growth medium was measured at times indicated on the horizontal axes. The results were normalised to Gluc activity in the growth media of cells transfected with the Gluc expression vector lacking an miRNA target site; the corresponding activity was set at 100% (shown on vertical axes). In all panels, open, black and grey columns represent normalised data for sponge design, perfect design and native design cassettes, respectively. Each column represents an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p<0.05) as determined by a Mann – Whitney U test.</p

Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells

Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

<p>Abstract</p> <p>Background</p> <p>The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems.</p> <p>Methods</p> <p>Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s).</p> <p>Results</p> <p>It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects.</p> <p>Conclusions</p> <p>Splice-switch technology, originally developed for genetic disease therapy, can also be used to control gene expression of viral vectors. This approach represents a novel, universal and powerful method for controlling gene expression, replication, viral spread and, by extension, virus-induced cytotoxic effects and can be used both for basic studies of virus infection and in virus-based gene- and anti-cancer therapy.</p