Activity and Tissue Distribution of Antisense Oligonucleotide CT102 Encapsulated with Cytidinyl/Cationic Lipid against Hepatocellular Carcinoma.

Insulin-like growth factor 1 receptor (IGF1R), a cell surface receptor with tyrosine kinase (TK) activity, has ligands abnormally expressed in acute leukemia, multiple myeloma, breast, prostate, cervical, and nonsmall cell lung cancers, Ewing's sarcoma, and other malignant tumors. IGF1R mediates the malignant proliferation, invasion, and metastasis of tumor cells through a variety of signal transduction pathways, and it is also involved in tumor angiogenesis and tumor cell antiapoptosis. In this study, the neutral cytidinyl lipid DNCA and cystine skeleton cationic lipid CLD from our laboratory could be optimized to encapsulate antisense oligonucleotide (ASO) CT102 to form stable and uniform Mix/CT102 nanoparticles (NPs), which could specifically target tumor cells that highly expressed IGF1R in vivo by intravenous administration. Compared with naked CT102, the lipid complex could promote the uptake and late apoptosis levels of HepG2 and Huh-7 cells, inhibiting cell proliferation efficiently. We also found that Mix/CT102 could enter nucleus in about 2 h, effectively downregulating the mRNA level of IGF1R. The in vivo efficacy experiment demonstrated that in the group that received the optimal dose of Mix/CT102, tumor volume was reduced 8-fold compared with the naked dose group. Meanwhile, in vivo distribution studies showed that the nanoparticles had a predominant accumulation capacity in liver tissue. These results indicated that clinicians can expect the Mix/CT102 nanocomposite to be very effective in reducing the dose and frequency of clinically administered CT102, thereby reducing the side effects of ASOs.

Modified ASO conjugates encapsulated with cytidinyl/cationic lipids exhibit more potent and longer-lasting anti-HCC effects.

Antisense oligonucleotides (ASOs) are a class of therapeutics targeting mRNAs or genes that have attracted much attention. However, effective delivery and optimal accumulation in target tissues in vivo are still challenging issues. CT102 is an ASO that targets IGF1R mRNA and induces cell apoptosis. Herein, a detailed exploration of the tissue distribution of ASOs delivered by liposomes was carried out. A formulation that resulted in increased hepatic accumulation was identified based on multiple intermolecular interactions between DCP (cytidinyl/cationic lipid DNCA/CLD and DSPE-PEG) and oligonucleotides, including hydrogen bonding, π-π stacking, and electrostatic interactions. The structurally optimized CT102s present a novel strategy for the treatment of hepatocellular carcinoma. The gapmer CT102MOE5 and conjugate Glu-CT102MOE5 showed superior antiproliferation and IGF1R mRNA suppression effects at 100 nM in vitro and achieved greater efficacy at a lower dose and administration frequency in vivo. Combined transcriptome and proteome analyses revealed that additional associated targets and functional regulations might simultaneously exist in ASO therapy. These results showed that a combination of lipid encapsulation and structural optimization in the delivery of oligonucleotide drugs has favorable prospects for clinical application.

Feasibility of cRGD conjugation at 5'-antisense strand of siRNA by phosphodiester linkage extension.

Small interfering RNAs (siRNAs) are widely studied for their highly specific gene silencing activity. However, obstacles remain to the clinical application of siRNAs. Attaching conjugates to siRNAs can improve their stability and broaden their application, and most functional conjugates of siRNAs locate at the 3'-terminus of the sense or antisense strand. In this work, we found that conjugating a group at the 5'-terminus of the antisense strand via phosphodiester was practicable, especially when the group was a flexible moiety such as an alkyl linker. When conjugating a bulky ligand, such as cRGD, the length of the 5'-phosphodiester linker between the ligand and the 5'-terminus of the antisense strand was the key in terms of RNA interference (RNAi). With a relative longer linker, the conjugates showed potency similar to siRNA. A highly efficient transfection system composed of a neutral cytidinyl lipid (DNCA) and a gemini-like cationic lipid (CLD) was employed to deliver siRNAs or their conjugates. The cRGD conjugates showed superior targeting delivery and antitumor efficacy in vivo and also selective cellular uptake in vitro. This unity of encapsulation and conjugation strategy may provide potential strategies for siRNA-based gene therapy.

siRNA Packaged with Neutral Cytidinyl/Cationic/PEG Lipids for Enhanced Antitumor Efficiency and Safety In Vitro and In Vivo.

The mutant BRAF gene is widely expressed in melanoma, and it acts as a suitable antitumor target. Small interference RNA (siRNA)-based therapy for BRAFV600E mRNA is, therefore, a path for melanoma clinical treatment owing to its high specificity. Although the U.S. Food and Drug Administration (FDA) approved the liver-target siRNA therapies, obstacles to siRNA tumor-targeted delivery still exist. Thus, an efficient tumor delivery system is an emergency. Here, we first report that the neutral cytidinyl lipid 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl)acetamide (DNCA) could encapsulate and transfer siRNA into the cytoplasm to induce gene silencing. Also, we sought the best formulation of DNCA/dioleoyl-3,3'-disulfanediylbis-[2-(2,6-diaminohexanamido)]propanoate (CLD)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEG2000-DSPE) for delivering siMB3, a siRNA for specific silencing of BRAFV600E mRNA. In the optimized formulation, the molar ratio of DNCA/CLD to a single nucleotide in siMB3 was 0.5/0.75/1 (the N/P ratio was about 3/1). Thanks to multiple forces including π-stacking, H-bonding, and electrostatic force between siRNA and lipids, the siRNA dose for effective gene silencing (85% knockdown) was reduced to 10 nM in vitro. Moreover, the siRNA lipoplexes with an additional 0.7% PEG-DSPE had a slightly negative charge and entered the cell mainly by caveolae-mediated endocytosis and macropinocytosis, avoiding degradation in the lysosome. These siRNA lipoplexes administrated through the tail vein also showed superior antitumor activity, with quite good safety and tissue distribution in vivo.