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Precondensed Plasmid DNA Enhances CAR‑T Cell Generation via Lipid Nanoparticles.

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DNA-condensing agents enhance lipid nanoparticle (LNP) delivery of plasmid DNA for chimeric antigen receptor (CAR) T-cell therapy. This method improves transfection efficiency and CAR expression, overcoming key challenges in non-viral vector development.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunotherapy

Background:

  • Chimeric antigen receptor (CAR) T-cell therapy offers personalized cancer treatment.
  • Viral vectors are currently used, but non-viral alternatives are sought for improved safety and efficacy.
  • Lipid nanoparticles (LNPs) are a promising non-viral delivery system for CAR T-cell therapy, but challenges remain in plasmid DNA (pDNA) encapsulation and expression.

Purpose of the Study:

  • To investigate the use of DNA-condensing agents in LNP manufacturing to improve pDNA delivery and expression.
  • To evaluate the impact of DNA condensation on LNP physicochemical properties and biological outcomes.
  • To assess the efficiency of LNP-mediated CAR gene delivery in relevant cell lines.

Main Methods:

  • Lipid nanoparticles (LNPs) were formulated using microfluidic technology with and without the P3000-Reagent (PR) condensing agent.
  • Physicochemical properties of LNPs were characterized.
  • Transfection efficiency and cellular localization of pDNA were assessed in HEK-293 cells using confocal microscopy.
  • Functional CAR gene delivery was evaluated in Jurkat T-lymphocytes upon multiple administrations.

Main Results:

  • Precondensation of pDNA with PR significantly increased LNP transfection efficiency.
  • Confocal microscopy revealed reduced lysosomal colocalization and enhanced nuclear localization of pDNA with PR treatment.
  • PR-treated LNPs demonstrated improved efficiency in delivering reporter and CAR-encoding plasmids to Jurkat cells, even with multiple administrations.

Conclusions:

  • Incorporating DNA-condensing agents like PR into LNP preparation is a viable strategy to overcome pDNA delivery and expression challenges.
  • This approach enhances the potential of LNPs for CAR T-cell therapy and other complex gene delivery applications.
  • The findings support the use of condensing agents for optimizing non-viral gene delivery systems, particularly for multi-administration protocols.