1. Volume 22, Issue 5, Pages 964-973 (May 2014)
Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery 
Cheng-Qiong Mao, Meng-Hua Xiong, Yang Liu, Song Shen, Xiao-Jiao Du, Xian-Zhu Yang, Shuang Dou, Pei-Zhuo Zhang, Jun Wang 
Molecular Therapy 
Volume 22, Issue 5, Pages (May 2014)
DOI: /mt
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
MNPsiCDK4-mediated synthetic lethal therapy for KRAS mutant non-small-cell lung carcinomas (NSCLCs). (a) A schematic view of synthetic lethality. Gene A and gene B are said to be synthetic lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes causes death.25 Regularly, a mutation of first gene A alone is essential to the development of cancer. In normal cells, both gene A and gene B are wild type; single mutation of gene B by stimulation has no effect on cell viability. However, in tumor cells, gene A has been mutant firstly for the tumor development. By synthetic lethal therapy, the mutation of second gene B (for example, via RNA interference or small molecule inhibiting gene B) would result in selective cytotoxicity of the tumor cells. (b) We designed an MNP delivery system self-assembled from amphiphilic block copolymers PCL29-PPEEA21 and PCL40-PEG45, which is capable of binding small interfering RNA (siRNA) and forming MNPsiCDK4 to specifically treat NSCLCs harboring mutant KRAS. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing siRNA targeting CDK4; PCL29-PPEEA21, poly(ε-caprolactone)-poly(2-aminoethylethylene phosphate); PCL40-PEG45, poly(ε-caprolactone)-poly(ethylene glycol).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
MNPsiCDK4 effectively transfects non-small-cell lung carcinoma (NSCLC) cell lines and human hepatocytes with small interfering RNA (siRNA). (a) Dynamic light scattering characterization of MNPs and transmission electronic microscopic images of MNPs. (b) Gel retardation assay of MNP/siRNA complexes at different N/P ratios. (c) MNPsiCDK4-mediated gene silencing in A549 cell line (mutant KRAS); H226 cell line (wild-type KRAS), and HL7702 cell line (wild-type KRAS) by real-time polymerase chain reaction at N/P = 10, siCDK4 = 100 nmol/l and N/P = 5, siCDK4 = 50, 100, and 200 nmol/l, respectively; and by western blotting at N/P = 5, siCDK4 = 100 and 200 nmol/l. The dose of siCDK4 for LiposiCDK4 was 50 nmol/l, and the dose of siN.C. depended on the maximum dose of siCDK4 at the same N/P ratio. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing siRNA targeting CDK4; mRNA, messenger RNA; PBS, phosphate-buffered saline; siN.C., negative control small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Effects of CDK4 knock down on the viability and proliferation of multiple cell lines. (a) Cell viability in the A549, H226, and HL7702 cell lines, 72 hours after transfection with MNPsiCDK4 at different concentrations. (b) Effects of CDK4 knock down on colony formation of the A549, H661, and HL7702 cell lines. Photographs of crystal violet-stained colonies are shown. (c,d) Multicolor competition assay 72 hours after transfection with MNPsiCDK4. KRAS mutant cells expressing green fluorescent protein (GFP) (A549-GFP) and KRAS wild-type cells (H226 or HL7702 cells) were mixed and transfected with siCDK4. The mutant and wild-type cell ratio at the end of the experiment was measured using FACS . If selective toxicity to mutant cells occurred rather than to wild-type cells, a reduced percentage of mutant cells would be detected. (e) The percentage of KRAS mutant cells in the mixture transfected with different formulations was normalized against that of a phosphate-buffered saline (PBS) control. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing small interfering RNA targeting CDK4; siN.C., negative control small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Antitumor growth by intravenous injection of various formulations. (a) Inhibition of A549 xenograft tumor growth by MNPsiCDK4. Intravenous injection started 12 days after s.c. injection of A549 cells. Doses of small interfering RNA (siRNA) and MNP for intravenous injection were 2 and 28.6 mg/kg per injection every 2 days, respectively. (b) Images of A549 xenograft tumors at the final time point of the treatment. (c) MNPsiCDK4 did not attenuate H226 xenograft tumor growth in vivo. Intravenous injection started 40 days after s.c. injection of H226 cells. The dose of administration was as same as that in the A549 xenograft tumor model. (d) Images of H226 xenograft tumors at the final time point of the treatment. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing siRNA targeting CDK4; PBS, phosphate-buffered saline; siN.C., negative control small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
MNPsiCDK4 mediated CDK4 expression knock down in vivo. (a) Expression level of CDK4 messenger RNA (mRNA) in A549 tumor tissue determined by quantitative real-time polymerase chain reaction at the final time point of the treatment (orange, phosphate-buffered saline (PBS); blue, MNP; pink, MNPsiN.C.; and purple, MNPsiCDK4). All the tumor samples (seven mice per group) were lysed for analysis. The histogram in upper right corner reports the average CDK4 mRNA expression level for each treatment. (b) KRAS protein expression levels and the reduction of CDK4 protein expression levels in A549 xenograft tumor tissue by MNPsiCDK4 administration at the final time point of the treatment. Two samples of each group were randomly chosen for detection. (c) Expression level of CDK4 mRNA in H226 tumor tissue at the final time point of the treatment. All the tumor samples (five mice per group) were lysed for analysis. (d) KRAS and CDK4 protein expression levels in H226 xenograft tumor tissue. Two samples of each group were randomly chosen for detection. (e) Hematoxylin and eosin (HE) (400×), CDK4 (400×), and Ki67 (400×) analyses of tumor tissues at the final time point of the treatment. Left: A549 tumor tissue, right: H226 tumor tissue. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing small interfering RNA targeting CDK4; siN.C., negative control small interfering RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions