Specific Delivery Of Phi29 Chimeric PRNA/siRNA Nanoparticles To Tumor Cells And Chemical Modification Of PRNA And Chimeric PRNA/siRNA

Bacillus subtilis bacterial phage phi29 DNA packaging RNA (pRNA), discovered by Prof. Peixuan Guo, was indispensible in phi29 viral assembly system. It has been well documented that pRNA molecule contains intermolecular interaction domain and a 5'/3' double helix domain. This two domains fold independently with each other. At one hand, within the intermolecular domain, the upper loop (right hand loop) and lower loop (left hand loop) are of the most featured structure. pRNA can form dimer through this intermolecular loop-loop interaction. At the other hand, replacement or insertion of the 5'/3' helical domain (sequences before 5'/3'23/97) with exogenous motif or sequence does not interfere with pRNA folding and dimer formation, making it possible to redesign pRNA as gene-targeting and delivery vehicles.We utilized pRNA as a vector and introduced siRNA sequence into 5'/3' helical domain to fabricate chimeric pRNA/siRNA, tarteting green fluorescent protein (GFP), firefly luciferase and survivin, respectively. To specifically deliver siRNA to target cells, we further developed a pRNA dimer, fabricated by folate-pRNA monomer for cell recognition and chimeric pRNA/siRNA monomer for gene silencing. Chimeric pRNA/siRNA monomers targeting GFP, luciferase and survivin, were synthesized, respectively, and were first tested for their gene silencing activity by transfection. After confirming the silencing efficacy of chimer pRNA/siRNA monomer, the fabricated folate-pRNA & pRNA/siRNA dimers were incubated with FR over-expressing MCF-7 and Hela cells, respectively. Real-time PCR, western blot, fluorescence microscopy as well as flow cytometry were used to analyze gene silencing effect.Since RNA is vulnerable to nuclease degradation, it is critical to make the RNA portion of the RNA-based drug stable in vivo or in the blood circulation system. Studies have demonstrated that chemical modification could generate stable RNA. We thus introduced 2'-F and 2'-NH2 modified dCTP and/or dUTP, as well as 2'-O-Me modified CTP and/or UTP through in vitro transcription to generate chemical modified pRNA and to achieve an improvement of thermal stability and bio-distribution of pRNA. Since non-modified pRNA can form dimers by intermolecular loop-loop interaction, chemical modified pRNA can be detected by TBE polyacrylamide gel electrophoresis (PAGE) for dimer formation to evaluate the modification effect on pRNA. Moreover, in vitro phi29 DNA packaging system, which is indispensible of pRNA and was reported the most efficient bacteriophage motor, can also be applied to evaluate the functional activity chemical modified pRNA. All of all the three modification strategies,2'-F modification was proved the most favorable strategy in retaining pRNA correct folding and dimer formation as well as enabling pRNA resistant to RNase and FBS degradation. Given that chimeric pRNA/siRNA was previously demonstrated capable of silencing specific gene, it could be stabilized for future therapeutic animal trail application by 2'-F modification by incorporation of 2'-F-dCTP and 2'-F-dUTP into chimeric pRNA/siRNA molecule. Folding and dimer formation of 2'-F chimeric pRNA/siRNA (GFP) were analyzed on TBE PAGE, and gene silencing effect were observed by fluorescent microscope for GFP expression. Further, to explore the mechanism of 2'-F chimeric pRNA/siRNA gene silencing effect,2'-F chimeric pRNA/siRNA (GFP) was tested by recombinant Dicer enzyme processing in vitro. The stability of 2'-F chimeric pRNA/siRNA was also tested by incubation of 2'-F chimeric pRNA/siRNA with RPMI-1640 cell culture medium containing 10% FBS. Taken together, we report three advantages of using pRNA as vehicles in gene silencing as well as drug delivery. First, pRNA can be redesigned to fabricate dimer nanoparticles capable of specific targeting and gene silencing. Second,2'-F-dCTP&dUTP modification can stabilize pRNA as well as chimeric pRNA/siRNA without interfering with their folding and dimer formation. Last, the nuclease resistant 2'-F chimeric pRNA/siRNA was active in gene silencing, making it a promising candidate for future in vivo therapeutic study. Objects:The bacteriophage phi29 DNA packaging RNA (pRNA) has been reported to have novel applications in nanotechnology and nanomedicine. Fusion of the pRNA with other therapeutic and conjugate with chemical compounds did not interfere with pRNA dimer formation or abolish pRNA correct folding. Previously, we found that MCF-7 and Hela cells expressed high levels of folate receptor. To specific deliver siRNA to tumor cells, we developed pRNA dimers, fabricated by folate-pRNA monomers for cell recognition and chimeric pRNA/siRNA monomers for gene silencing. These folate-pRNA & pRNA/siRNA dimers were incubated with MCF-7 and Hela cells, respectively, and analyzed for their effect on down-regulation of target gene expression. To improve the thermal stability and bio-distribution of pRNA particle both in vitro and in vivo, we constructed chemical modified pRNA nanoparticles by introducing 2'-F and 2'-NH2 modified dCTP and/or dUTP, as well as 2'-O-Me modified CTP and/or UTP through in vitro transcription. Chemical modified pRNAs were examined for dimer formation, in vitro phi29 DNA assembly activity, and stability in RNase A or FBS. Based on chemical medication result,2'-F-dCTP&dUTP were incorporated into chimeric pRNA/siRNA molecule to generate nuclease resisitant chimeric pRNA/siRNA by in vitro transcription. The 2'-F chimeic pRNA/siRNA were further tested for dimer assemble activity, gene silencing effect, recombinant Dicer enzyme digestion profile as well as stability in cell culture medium containing 10% FBS.Methods:①pRNAs were prepared as described. folate-pRNA & pRNA/siRNA dimer was prepared by mixing same molar amount of folate-pRNA Ba' and pRNA Ab' /siRNA (GFP, luciferase or survivin) with the existence of 5 mM Mg2+.②Cells were seeded in a 96-well plate in folate-free cell culture medium. After twice rinse with PBS containing MgCl2, the premixed folate-pRNA & pRNA/siRNA dimer (GFP, luciferase and survivin) was then added to the cells and incubated with cells for 3h at 37℃. RNase inhibitor was added to the binding buffer. After incubation, pEGFP-N1, PGL3 and PRL-TK plasmids were introduced into the cells by Lipofectamine 2000. Cells were further incubated for 24 or 48 hr. GFP expression, firely luciferase activity and survivin knockdown were measured respectively. The expression of GFP was detected by fluorescent microscope and flow cytometry. Luciferase activities were measured in a dual reporter assay system 1 day after transfection. For survivin knockdown assay, cells were transfected with pRNA/siRNA (survivin) in 24-well plates. Expression of survivin and apoptosis of tumor cells were detected by Real-Time PCR, western blot and flow cytometry.③To generate nuclease resistant pRNA, construction of 2'-F,2'-NH2 and 2'-O-Me modified pRNA nanoparticles was performed by incorporating 2'-F or 2'-NH2 modified dCTP and/or dUTP, or 2'-O-Me modified CTP and/or UTP into pRNA molecules. It is crucial that the chemical modified pRNA still has the competency to assemble into dimers, trimers or hexamers. Dimer formations by each modified pRNAs were detected by TBE PAGE gel and phi29 DNA assembly system was applied to examine functional activity of modified pRNA.④To construct stable chimeric pRNA/siRNA,2'-F-dCTP and 2'-F-dUTP were introduced through in vitro transcription into 2'-F chimeric pRNA Ab'/siRNA. TBE PAGE assay was used to examine folding, and dimer formation of chimeric pRNA/siRNA monomer with its complementary pRNA Ba' monomer. Transfection of 2'-F chimeric pRNA/siRNA by lipofectamine 2000 was taken to evaluate the gene silencing effect on KB cells. The expression of GFP expression was observed by fluorescent microscope. Dicer enzyme processing was performed by incubation of 5'end labeled [γ-32P] 2'-F chimeric pRNA/siRNA with recombinant Dicer and the mixture were then analyzed by urea PAGE and visualized by film exposure. Finally, stability assay was carried out by treatment of 2'-F chimeric pRNA/siRNA with cell culture medium RPMI-1640 containing 10% FBS and then visualized by urea PAGE gel stained with ethidium bromide.Results:①Our previous study indicated that MCF-7 and Hela cells highly express folate receptors. Specific knockdown of the GFP and firefly luciferase expression by folate-pRNA & pRNA/siRNA dimers in MCF-7 and Hela cells, compared with controls, suggesting the gene silencing effect was achieved by folate receptor-mediated internalization of chimeric pRNA/siRNA in the absence of transfection reagents. After tumor cells were co-incubated with the folate-pRNA & pRNA/siRNA (survivin) dimer, knockdown effect of survivin expression in tumor cells were detected at the mRNA level by Real time-PCR and at the protein level by western blotting. Cell apoptosis was analyzed by flow cytometry assay, with Annexin V-FITC and PI staining. The data suggests that the folate-pRNA & pRNA/siRNA (survivin) dimer could specifically enter into MCF-7 and Hela cells, resulting in the significant inhibition of gene transcription and protein expression of survivin and leading to cell apoptosis.②To enhance the stability of the pRNA nanoparticle both in vitro and in vivo, we constructed chemical modified pRNA by introducing 2'-F,2'-NH2, and 2'-O-Me modified CTP and/or UTP through in vitro transcription. TBE PAGE showed that 2'-F modification of pRNA by 2'-F-dCTP and 2'-F-dUTP had retained pRNA correct folding and dimer formation ability. In vitro phi29 DNA assemble assay showed 2'-F pRNA had viral assembly activity of 106 pfm/ml, of which was slightly lower compared with non-modified pRNA with 107 pfin/ml activity, but still functionally active. However,2'-NH2 modification blocked pRNA correct folding and dimer formation and subsequent dramatic decrease of viral assembly activity or totally loss of activity. Whereas,2'-O-Me modification data indicated that 2'-O-Me-UTP was difficult to be recognized by RNA polymerase Y639F, thus 2'-O-Me-CTP modified pRNA was obtained, of which activity was low. In conclusion, the chemical modification data suggested that 2'-F modification, compared with 2'-NH2 and 2'-O-Me, was the favorite strategy that had reserved pRNA functional activity and was suitable for stabilize pRNA, while not causing serious impact on pRNA folding, dimer formation, and subsequent assembly activity.③To stabilize the chimeric pRNA/siRNA for future in vivo animal trial application,2'-F-dCTP and 2'-F-dUTP modification was introduced into chimeric pRNA/siRNA (GFP) molecule by in vitro transcription. TBE PAGE showed dimer formation of 2'-F chimeric pRNA Ab'/siRNA with its complementary pRNA Ba' monomer. Co-transfection of 2'-F pRNA/siRNA (GFP) with GFP-expressing plasmid EGFP-N1 to KB cells revealed by fluorescent microscope that 2'-F modified pRNA/siRNA (GFP) effectively inhibited GFP gene expression and exhibited comparable inhibitory effects with none-modified pRNA/siRNA (GFP). Dicer enzyme processing data showed by urea PAGE that 2'-F-dUTP modified pRNA/siRNA (GFP) released a small RNA band close to 22 nt control RNA. Whereas,2'-F-dCTP and 2'-F-dCTP&dUTP modified pRNA/siRNA (GFP) showed similar size of processed siRNA to each other, which shifted slower than that of 2'-F-dUTP modified pRNA/siRNA (GFP) and 22 nt control RNA. The processing results suggested that the dicer enzyme might recognize and cleave specific nucleotides inside the chimeric pRNA/siRNA, which could not possibly be cleaved after 2'-F modification, especially for 2'-F-dCTP modification. Conclusion:Taken together, we report systemically for the first time that the pRNA dimer fabricated by folate-pRNA and chimeric pRNA/siRNA could specifically deliver therapeutic molecule siRNA and down-regulate target gene expression in MCF-7 and Hela cells, which exhibited high expression of folate receptors. The folate-pRNA & pRNA/siRNA targeting GFP, luciferase, as well survivin was tested for their gene silencing effect and data suggested the specific knockdown of the GFP, luciferase and survivin expression by fluorescence microscopy, Real-Time PCR, western bolt and flow cytometry, respectively. Chemical modification of pRNA indicated that 2'-F modification retained pRNA folding and dimer formation ability, as well as its phi29 DNA assembly activity. To stabilize chimeric pRNA/siRNA for future therapeutic application in animal trails,2'-F modification was introduced into chimeric pRNA/siRNA molecule. Dimer formation assay and gene silencing data supported that 2'-F chimeri pRNA/siRNA (GFP) was of correct folding and capable of gene silencing. Recombinant Dicer processing result suggest 2'-F chimeric pRNA/siRNA (GFP) can be processed and release siRNA with a smaller size, which might due to some specific Dicer recognized or processing nucleotide could not possibly be cleaved after 2'-F modification.