Calcium–MicroRNA Complex-Functionalized Nanotubular Implant Surface for Highly Efficient Transfection and Enhanced Osteogenesis of Mesenchymal Stem Cells

Controlling mesenchymal stem cell (MSC) differentiation by RNA interference (RNAi) is a promising approach for next-generation regenerative medicine. However, efficient delivery of RNAi therapeutics is still a limiting factor. In this study, we have developed a simple, biocompatible, and highly effective delivery method of small RNA therapeutics into human MSCs (hMSCs) from an implant surface by calcium ions. First, we demonstrated that simple Ca/siRNA targeting green fluorescent protein (GFP) nanocomplexes were able to efficiently silence GFP in GFP-expressing hMSCs with adequate Ca²⁺ concentration (>5 mM). In addition, a single transfection could obtain a long-lasting silencing effect for more than 2 weeks. All three of the main endocytosis pathways (clathrin- and caveolin-mediated endocytosis and macropinocytosis) were involved in the internalization of the Ca/siRNA complexes by MSCs, and macropinocytosis plays the most dominant role. Furthermore, the Ca/siRNA complexes could be efficiently loaded onto the titanium implant surface when pretreated with anodization to create a nanotube (NT) layer. Because of the hydrophilic property of the NT surface, the Ca/siRNA was quickly loaded (less than 4 h) with high efficiency (nearly 100%), forming an even amorphous coating. The Ca/siRNA-coated NT surface showed an initial burst release of 80% of the siRNA complexes over 2 h, which is adequate to achieve robust gene silencing of attached hMSCs. To demonstrate the therapeutic potential of our Ca/siRNA coating technology, Ca/antimiR-138 complexes were loaded on to the NT surface, which strongly enhanced the osteogenic differentiation of hMSCs. In conclusion, our findings suggest that Ca²⁺ is an effective and biocompatible carrier to deliver small RNA therapeutics into hMSCs, both in solution and from functionalized surfaces, which provides a novel approach to control the MSC differentiation and tissue regeneration

Functional analyses of RRE with changes at positions 40 and 45 in lymphoid cells.

<p>The lymphoid cell line MT-4 was co-electroporated with the NL4-3 hemigenomic plasmids p83-2 and with the p83-10 constructed variants, which only differed in their RRE region. A) Fold increase of cytosolic unspliced RNA in infected cells. Levels of unspliced RNA in the cytoplasmic fractions were determined by qRT-PCR. The fold change was calculated by the relative quantitation method 2(^−ΔΔCt). GAPDH was used for normalization and RREWT (40Q-45L) as a calibrator. Data (mean +/− SEM) is derived from three independent experiments, with triplicate samples in each PCR. B) Quantification of the transcriptional activity per infected cell. The ratio between the levels of unspliced HIV RNA and total HIV DNA (2(^−ΔΔCt) Unspliced HIV-1 RNA/2(^−ΔΔCt) Total HIV-1 DNA) was calculated. Total HIV DNA content was determined in cells by qPCR using the same approach as described with the unspliced RNA levels. Data (mean +/− SEM) is derived from three independent experiments, with triplicate samples in each PCR. C) P24 protein production in cell-free supernatants. The p24 present in the supernatant of the cultures was quantified by ELISA from the same time-point that the RNA and DNA levels were determined. Data (mean +/− SEM) is derived from two independent experiments. D) Normalized p24 protein production. Raw p24 values were normalized to copies of total HIV-1 DNA to correct for differences in electroporation efficiency and gp41 function. Data (mean +/− SEM) is derived from two independent experiments.</p

Functional Analyses Reveal Extensive RRE Plasticity in Primary HIV-1 Sequences Selected under Selective Pressure

<div><p>Background</p><p>HIV-1 Rev response element (RRE) is a functional region of viral RNA lying immediately downstream to the junction of gp120 and gp41 in the <i>env</i> coding sequence. The RRE is essential for HIV replication and binds with the Rev protein to facilitate the export of viral mRNA from nucleus to cytoplasm. It has been suggested that changes in the predicted secondary structure of primary RRE sequences impact the function of the RREs; however, functional assays have not yet been performed. The aim of this study was to characterize the genetic, structural and functional variation in the RRE primary sequences selected <i>in vivo</i> by Enfuvirtide pressure.</p><p>Results</p><p>Multiple RRE variants were obtained from viruses isolated from patients who failed an Enfuvirtide-containing regimen. Different alterations were observed in the predicted RRE secondary structures, with the abrogation of the primary Rev binding site in one of the variants. In spite of this, most of the RRE variants were able to bind Rev and promote the cytoplasmic export of the viral mRNAs with equivalent efficiency in a cell-based assay. Only RRE45 and RRE40-45 showed an impaired ability to bind Rev in a gel-shift binding assay. Unexpectedly, this impairment was not reflected in functional capacity when RNA export was evaluated using a reporter assay, or during virus replication in lymphoid cells, suggesting that <i>in vivo</i> the RRE would be highly malleable.</p><p>Conclusions</p><p>The Rev-RRE functionality is unaffected in RRE variants selected in patients failing an ENF-containing regimen. Our data show that the current understanding of the Rev-RRE complex structure does not suffice and fails to rationally predict the function of naturally occurring RRE mutants. Therefore, this data should be taken into account in the development of antiviral agents that target the RRE-Rev complex.</p></div

<i>In vitro</i> Rev-RRE binding assay.

<p>Electro-mobility shift assay (EMSA) in the absence or in the presence of 20 ng, 40 ng or 80 ng of Rev protein with the different RRE RNA variants (RREWT, sRRE40, sRRE45 and RRE40-45). The reaction products were separated in a polyacrylamide gel and the quantification of the gel shifts are displayed.</p

Predicted secondary structures of patient-derived RRE variants.

<p>The prediction of the secondary structure of our RRE sequences was generated including the 329-nucleotide sequence of the patient-derived RREs in the RNA Fold Web server. The five well-defined stem-loop structures, including the branched stem-loop IIB that is critical for the binding of the Rev protein, were identified for most of the sequences. A) Predicted secondary structure of RRE from patient 10. Predicted secondary structure of the complete RRE of a BL clone from patient 10 is shown. The nucleotides encoding the amino acid changes associated with ENF resistance are underlined and a dotted box encloses the high-affinity Rev binding site. Stems loops II-III-IV and V for representative samples for each substitution present in Patent 10 are shown. Nucleotide changes present in the RRE variants with regard to the BL clone are marked with filled red circles. B) Representative RRE structures obtained from samples of patient 5.</p

Alignment of patient-derived RRE variants used for functional analysis.

<p>Multiple nucleotide alignment of the full-length patient-derived RRE variants used for prediction of secondary structures and functional analyses. Several full-length RREs derived from two patients (patient 10: P10; and patient 5: P5) were amplified from plasma samples, cloned and sequenced. Boxes highlight the nucleotides coding ENF resistance mutations G36V/D, V38A and N43D for patient 10; and Q40H and L45M for patient 5. The characteristic five stem-loop regions of the RRE are identified on top of the sequence. Variants were designated according to the patient number, amino acid change in gp41 and clone number. The shaded areas indicate the reported high-affinity binding site of Rev located in the stem II. The NL4-3 RRE sequence is included for comparison.</p

Predicted secondary structures of RRE variants constructed by site-directed mutagenesis.

<p>RRE variants with nucleotide substitutions at position 40 sRRE40 (Q40H), at position 45 sRRE45 (L45M) or without any, WT (40Q-45L), were generated by site-directed mutagenesis using as a template a RRE40-45 clone (Q40H-L45M). The sequences of these constructed clones were subjected to RNA fold analyses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106299#pone-0106299-g002" target="_blank">Figure 2</a>. Nucleotides that were substituted are highlighted in the filled red circles.</p

Rev-dependent RNA transport.

<p>293T cells were co-transfected with the constructed RRE variants, pDM628 or pDM628ΔRRE with or without pCMV-Rev. The export levels of the different variants were quantified by luminescence and corrected by the background signal from the luminometer noise or due to Rev-independent transport. This correction was performed to each sample by subtracting the luminescence that was measured when the cells were transfected without Rev (replaced with pcDNA 3.0). And finally, corrected luminescence values were calculated as the fold-change increase, which was performed by dividing the corrected luminescence of each plasmid by the corrected luminescence of the pDM628 plasmid. A) Rev-RRE mediated export from RNA variants containing changes at positions 36, 38 and 43, evaluated in the presence of Rev (ratio 1:5, Rev:RRE). B) Cytoplasmic export of RRE variants with changes at positions 40 and 45. Rev-dependent transport of the pDM628-based RRE variants: WT (40Q-45L), sRRE40 (Q40H), sRRE45 (L45M) and the double mutant RRE40-45 (Q40H-L45M); in the presence of three different concentrations of pCMV-Rev (200 ng, 20 ng or 2 ng per well). Data represent the mean +/− SEM of 3 independent experiments performed in triplicate transfections.</p

<i>In silico</i> analysis of the sequences from the loop-selections.

<p>Consensuses secondary structures of high and low functionality loops predicted by RNAforester <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043095#pone.0043095-Hochsmann1" target="_blank">[20]</a> based on MFold3.2 generated structures after the 1^st selection round (<b>A</b>) and 2^nd selection round (<b>B</b>). Loops of 7 nt, 9 nt and 11 nt loop libraries are shown from left to right. The figure shown uses standard RNAforester output settings: Each base position is represented by a square where the corners represent the four bases with a dot. The size of the dot represents the frequency of the particular base; colour code: Red-A, yellow-U, green-C, blue-G, black circle: the frequency of a gap is proportional to a black circle growing at the centre of the square. Bases or base pair bonds that have a frequency of one hundred percent are drawn in red color. The blue arrow indicates the last base pair of the duplex stem region. Sequences displaying a stretch of 4 or more uracils, have been removed to avoid contribution from transcripts terminating prematurely <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043095#pone.0043095-Mathews1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043095#pone.0043095-Zuker1" target="_blank">[19]</a>. (<b>C</b>). Evaluating shRNA processing by northern blotting. shRNA RNA vectors harboring shRNA targeting sequence #1 and the indicated loops were transiently transfected into H1299 cells and shRNA processing were evaluated by 15% denaturing PAGE and northern blotting using a 19-mer probe against the processed eGFP antisense strand of the shRNA. Both the mature 51-nt and processed 21-nt RNA species are identified for the efficient loop where no processing is seem for the inefficient loop.</p

Testing loops in context of different stem-sequences.

<p>The shRNA cassettes were either introduced stably using retroviral vector (<b>A</b>) or transiently from a plasmid co-transfections with a firefly luciferase reporter construct containing the eGFP target sequence (<b>B</b>). eGFP levels were measured by Flow cytometry. Asterisk denotes the control shRNA containing the loop previously published by Brummelkamp et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043095#pone.0043095-Brummelkamp1" target="_blank">[11]</a>.</p