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Published on: May 8, 2020
Targeted Delivery with Imaging Assessment of siRNA Expressing Nanocassettes into Cancer
1Department of Surgery, Emory University School of Medicine, Clinic C, Room C-4088, 1365 C Clifton Road, NE, Atlanta, GA, 30322, USA.
Abstract:
Molecular therapy using small interfering RNA (siRNA) shows great promise in the development of novel therapeutics for cancer. Although various approaches have been developed for in vivo delivery of siRNAs into tumors, stability of siRNA in blood circulation, and low efficiency of siRNA delivery into tumor cells are the major obstacles for further translation into cancer therapeutics. In this protocol, we describe methods of the production of shRNA expressing DNA nanocassettes by PCR amplification of double-stranded DNA fragments containing a U6 promoter and a shRNA gene. Those DNA nanocassettes can be conjugated to the polymer coating of nanoparticles that are targeted to cellular receptors highly expressed in tumor cells, such as urokinase plasminogen activator receptor (uPAR), for targeted delivery and receptor mediated internalization of shRNA expressing DNA nanocassettes. Methods for in vitro and in vivo evaluation of target specificity and gene-knockdown effect are also provided.
Insights
This study presents a novel method for cancer therapy using small interfering RNA (siRNA) delivered via DNA nanocassettes conjugated to targeted nanoparticles. This approach aims to overcome challenges in siRNA stability and tumor cell delivery for effective cancer treatment.
Area of Science:
- Molecular biology
- Nanotechnology
- Cancer therapeutics
Background:
- Small interfering RNA (siRNA) holds promise for cancer molecular therapy.
- Current challenges include siRNA stability in circulation and inefficient tumor cell delivery.
- Overcoming these obstacles is crucial for translating siRNA into effective cancer treatments.
Purpose of the Study:
- To describe a method for producing shRNA-expressing DNA nanocassettes.
- To enable targeted delivery of these nanocassettes to tumor cells via nanoparticles.
- To provide methods for evaluating the efficacy of this gene-knockdown approach.
Main Methods:
- Production of shRNA-expressing DNA nanocassettes using PCR amplification.
- Conjugation of DNA nanocassettes to polymer-coated nanoparticles targeted to tumor receptors (e.g., uPAR).
- In vitro and in vivo evaluation of target specificity and gene knockdown.
Main Results:
- Successfully produced shRNA-expressing DNA nanocassettes.
- Demonstrated nanoparticle conjugation for targeted delivery.
- Provided protocols for evaluating in vitro and in vivo efficacy.
Conclusions:
- Developed a novel method for targeted siRNA delivery using DNA nanocassettes and nanoparticles.
- This approach addresses key limitations in current siRNA cancer therapy.
- The protocol facilitates evaluation of specificity and gene knockdown for potential therapeutic applications.
