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TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents
Published on: June 2, 2016
A polymer library approach to suicide gene therapy for cancer
Daniel G Anderson1, Weidan Peng, Akin Akinc
1Department of Chemical Engineering and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.
Insights
Researchers developed a new polymer, C32, for efficient and safe cancer gene therapy. This nonviral DNA vector effectively targets tumors with minimal toxicity, showing promise for localized cancer treatment.
Area of Science:
- Biotechnology
- Polymer Chemistry
- Cancer Gene Therapy
Background:
- Effective cancer gene therapy requires efficient tumor DNA delivery, minimal toxicity, and restricted expression in healthy tissues.
- Nonviral DNA vectors are crucial for safe and targeted gene delivery in cancer treatment.
Purpose of the Study:
- To develop and evaluate a novel degradable poly(beta-amino ester) polymer (C32) as a nonviral DNA vector for cancer gene therapy.
- To assess the in vitro and in vivo performance of C32 in terms of transfection efficiency, cytotoxicity, and tumor targeting.
Main Methods:
- Screened a library of over 500 poly(beta-amino esters) using high-throughput methods for in vitro transfection efficiency and cytotoxicity.
- Tested the lead polymer, C32, in mice for toxicity and DNA delivery via intratumoral and intramuscular injection.
- Delivered a DNA construct encoding diphtheria toxin A (DT-A) using C32 to LNCaP prostate cancer xenografts.
Main Results:
- C32 demonstrated significantly higher intratumoral DNA delivery compared to polyethyleneimine (jetPEI) and naked DNA.
- Intramuscular injection showed minimal transfection with C32 in healthy muscle tissue, unlike naked DNA or polyethyleneimine.
- Polyethyleneimine induced significant local toxicity after intramuscular injection, whereas C32 showed no toxicity.
- C32-mediated delivery of DT-A DNA suppressed tumor growth and led to a 40% regression in LNCaP xenografts.
Conclusions:
- C32 is a promising nonviral vector for local cancer gene therapy due to its high tumor transfection efficiency, low toxicity, and minimal off-target transfection in healthy tissues.
- The developed polymer offers a potential new strategy for localized cancer treatment by effectively delivering therapeutic genes to tumors.
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