Specific Delivery of Therapeutic RNAs to Cancer Cells via the Dimerization Mechanism of phi29 Motor pRNA

Abstract

The application of small RNA in therapy has been hindered by the lack of an efficient and safe delivery system to target specific cells. Packaging RNA (pRNA), part of the DNA-packaging motor of bacteriophage phi29(φ29), was manipulated by RNA nanotechnology to make chimeric RNAs that form dimers via interlocking right-and left-hand loops. Fusing pRNA with receptor-binding RNA aptamer, folate, small interfering RNA (siRNA), ribozyme, or another chemical group did not disturb dimer formation or interfere with the function of the inserted moieties. Incubation of cancer cells with the pRNA dimer, one subunit of which harbored the receptor-binding moiety and the other harboring the gene-silencing molecule, resulted in their binding and entry into the cells, and subsequent silencing of anti/proapoptotic genes. The chimeric pRNA complex was found to be processed into functional double-stranded siRNA by Dicer (RNA-specific endonuclease). Animal trials confirmed the suppression of tumorigenicity of cancer cells by ex vivo delivery. It has been reported [Shu, D., Moll, W.-D., Deng, Z., Mao, C., and Guo, P. (2004). Nano Lett. 4:1717Lett. 4: -1724 that RNA can be used as a building block for bottom-up assembly in nanotechnology. The assembly of protein-free 25-nm RNA nanoparticles reported here will allow for repeated long-term administration and avoid the problems of short retention time of small molecules and the difficulties in the delivery of particles larger than 100 nm. OVERVIEW SUMMARYThe successful use of small RNA for therapeutic purposes requires a safe and efficient delivery system capable of targeting specific cells. φ29 motor RNA (pRNA) was manipulated to produce chimeric RNAs that formed dimers through connection by right-and left-hand loops. Fusion of the pRNA with a variety of therapeutic and chemical compounds did not impede the formation of dimers or interfere with moiety function. Incubation of cells with dimer, one subunit of which carried a gene-silencing molecule and the other a receptor-binding moiety, resulted in successful binding, entry, and silencing of apoptotic genes. Animal trials further confirmed suppression of the tumorigenicity of cancer cells by ex vivo delivery. The dimeric RNA chimera was processed into double-stranded small interfering RNA (siRNA) by Dicer. These protein-free 25-nm nanoparticles will allow for repeated and long-term administration escaping immunoresponse and avoid the short retention time of smaller molecules and the undeliverability of larger molecules.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

“…A chimeric pRNA/siRNA monomer was constructed by replacing the double-stranded helical region of pRNA with siRNA sequences without affecting the gene silencing function and the pRNA secondary structure. 21,22 The deliverable folate containing nanoparticles was conjugated by mixing equal molar amounts of folate-pRNA (B-a 0 ) with a chimeric pRNA/siRNA (A-b 0 ) via the interaction of the interlocking loops ( Figure 3 Entry of nanoparticles containing both folic-pRNA and siRNA chimera to nasopharyngeal carcinoma cells To determine whether the folate moiety on the chimeric pRNA dimer could mediate the entry of the complex into KB cells, a chimeric siRNA complex was constructed for specific gene silencing. RNA dimer (1.75 mM), containing both folate and siRNA against firefly luciferase, was incubated with, rather than transfected into, KB cells.…”

“…16,21,22 In this study, a chimeric pRNA/siRNA targeting firefly or renilla luciferase was constructed, and the silencing efficiency was tested by transient transfection. The chimeric siRNA construct suppressed its target gene specifically and efficiently as demonstrated by a Dual reporter assay, in which the expression To determine whether the knockdown effects shown above were siRNA-specific, chimeric pRNA/siRNA monomers or dimers were treated with cell lysate or recombinant purified Dicer ( Figure 5), which is known for its unique function in processing long doublestranded RNA into 22-bp siRNA.…”

“…[18][19][20] Recently, we found that phi29 pRNA can be used as a carrier for the construction of RNA nanoparticles to deliver therapeutic RNAs such as siRNAs and/or ribozymes to specific cancer cells. 12,21,22 In this report, we described the methods for the synthesis and purification of folate-adenosine 5 0 -monophosphate (AMP), the procedure for the construction of folatepRNA, and the test of the purity and function of the folate-containing products. The potential of siRNA in gene therapy mediated by folate was also investigated in animal trials.…”

Aptamers in Immunotherapy

“…In 1998, two aptamers were described that were specific to the human and rat homolog that bind the receptor with high affinity (Kraus et al, 1998). The ligand targeting the rat receptor was shown to inhibit CD4 function in vitro, whereas the ligand specific to the human receptor is used as a targeting moiety to fluorescently label CD4+ cells (Davis et al, 1998) and deliver cargo (including siRNA) to cells expressing the receptor (Guo et al, 2005).…”

Bottom-up assembly of RNA nanoparticles containing phi29 motor pRNA to silence the asthma STAT5b gene

“…Therefore, the 5ꞌ/3ꞌ-proximate double-stranded helical region of the pRNA can be redesigned to carry additional sequences without altering its secondary structure or intermolecular interactions. On the basis of these characteristics, incubation of cells with pRNA dimmers, one subunit of which carried siRNA and the other, a receptor-binding moiety, resulted in successful binding, entry, and silencing of target genes (Guo et al, 2005;Zhang et al, 2009;Zhou et al, 2011).…”