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Updated: May 5, 2026
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Published on: December 21, 2011
Systemic dual delivery of a tumor-inhibiting plasmid expressing p16 and shRaf
Qixin Leng1, Zuha Imtiyaz1, A James Mixson1
1Department of Pathology, University of Maryland School of Medicine, 10 S. Pine St., University of Maryland, Baltimore, MD, 21201, USA.
Abstract:
Efforts to target tumors in vivo with high-molecular-weight plasmids have been hindered by inefficient delivery. Moreover, while siRNA and mRNA have targets in the cytosol, plasmid-based therapy must reach the nucleus to express its products. Consequently, many groups have abandoned robust self-amplifying plasmid-based therapies in favor of siRNA or mRNA. An efficient delivery system for plasmids could transform breast cancer treatment and represent an important step toward successful "gene" therapy in the clinic. Although selecting a tumor suppressor gene replacement, antiangiogenic gene expression, or shRNA expressed by plasmids, along with their synergy with adjuvant therapy, is important, these strategies are limited without an effective delivery system to the tumor. This report investigates whether polymeric carriers for plasmid-based therapy inhibit the growth of MDA-MB-231 cells. These triple-negative breast cancer cells display an enhanced Ras/Raf-1/ERK (ERK) pathway and lack the wild-type tumor suppressor p16. Using several polymeric carriers, we found that the plasmid expressing p16 and shRaf effectively suppressed tumor cells in vitro. Notably, despite low amounts in the polyplex, the histidine-lysine (HK) peptide carrier showed increased gene expression in the tumor xenografts compared to normal tissues. In contrast to tumor xenografts, no toxicity in normal tissues was observed with the p16shRaf therapy. The HK plasmid-based polyplex markedly reduced tumor size by approximately 75 % compared to untreated controls. The antitumor effect of the p16shRaf-containing polyplex was due to increased apoptosis and decreased mitosis in tumor xenograft cells.
Insights
Polymeric carriers effectively deliver plasmid-based gene therapy to triple-negative breast cancer cells. This approach significantly reduces tumor size by promoting apoptosis and decreasing mitosis, offering a promising new avenue for cancer treatment.
Area of Science:
- Oncology
- Gene Therapy
- Nanomedicine
Background:
- Plasmid-based gene therapies face challenges with in vivo tumor targeting and inefficient delivery to the cell nucleus.
- Current limitations in delivery systems have led researchers to favor siRNA or mRNA therapies over robust plasmid-based approaches.
- Triple-negative breast cancer (TNBC) cells, like MDA-MB-231, often exhibit dysregulated signaling pathways (e.g., Ras/Raf-1/ERK) and lack tumor suppressors (e.g., p16).
Purpose of the Study:
- To investigate the efficacy of polymeric carriers for delivering plasmid-based therapy targeting MDA-MB-231 triple-negative breast cancer cells.
- To evaluate the potential of plasmid-based delivery of p16 (tumor suppressor) and shRaf (short hairpin RNA targeting Raf-1) for cancer treatment.
- To assess the safety and tumor-specific gene expression of plasmid-loaded polymeric carriers in vivo.
Main Methods:
- Utilized several polymeric carriers to formulate polyplexes with plasmids encoding p16 and shRaf.
- Tested the efficacy of these polyplexes against MDA-MB-231 cells in vitro.
- Administered the histidine-lysine (HK) peptide-based polyplexes to tumor xenografts in vivo to assess gene expression, tumor size reduction, and tissue toxicity.
Main Results:
- Plasmid expressing p16 and shRaf effectively suppressed MDA-MB-231 tumor cells in vitro.
- The histidine-lysine (HK) peptide carrier demonstrated enhanced gene expression in tumor xenografts compared to normal tissues, despite low polyplex amounts.
- HK polyplex treatment resulted in a significant reduction of tumor size (approximately 75%) with no observed toxicity in normal tissues.
- The antitumor effect was attributed to increased apoptosis and decreased mitosis within the tumor xenograft cells.
Conclusions:
- Polymeric carriers, particularly the HK peptide-based polyplex, represent an effective delivery system for plasmid-based gene therapy in triple-negative breast cancer.
- This strategy successfully targets tumor cells, leading to significant tumor growth inhibition and enhanced apoptosis.
- The HK polyplex shows promise for safe and effective in vivo gene therapy, overcoming previous delivery limitations and offering a potential new treatment modality for breast cancer.
