Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Nanomedicine

Background:

• Triplex gene editing utilizes peptide nucleic acid (PNA) binding to chromosomal targets to facilitate site-specific recombination with single-strand DNA (ssDNA) donor templates for gene correction.
• Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles offer a potential delivery method for PNA and ssDNA donor templates in gene therapy applications.

Purpose of the Study:

• To evaluate the efficacy of codelivered PNA and ssDNA donor templates via PLGA nanoparticles for correcting sickle cell disease and x-linked severe combined immunodeficiency (SCID).
• To identify and characterize potential artifacts in PNA-mediated gene editing assessments.

Main Methods:

• Encapsulation of PNA and ssDNA donor templates within PLGA nanoparticles for codelivery.
• In vitro and in vivo assessment of gene correction in models of sickle cell disease and x-linked SCID.
• Polymerase Chain Reaction (PCR) artifact analysis to detect PNA-ssDNA aggregation.

Main Results:

• Codelivery of PNA and ssDNA donor templates, but not individual agents, resulted in varying degrees of aggregation.
• This aggregation led to reproducible false-positive PCR signals in vitro, indicating artifactual gene correction.
• PNA sequences showing high in vitro correction did not demonstrate efficacy in vivo, underscoring the artifact's impact.

Conclusions:

• PNA-ssDNA aggregation is a significant source of false-positive results in PNA-mediated gene editing assessments.
• Eliminating ssDNA donor template carryover and interrogating aggregation are critical for validating gene editing technologies.
• This finding emphasizes the need for rigorous validation of PNA-mediated gene editing approaches in preclinical models.