Systemic gene transfer of polyethylenimine (PEI)–plasmid DNA complexes to neonatal mice

Purpose: Nonviral vectors have rarely been tested in neonatal mice because of poor delivery efficiency, yet early gene therapy could prevent irreversible disease, requires lower vector doses due to small body size, and may avoid immune reactions before immune maturation. Viral vectors carry risks such as insertional mutagenesis and immunogenicity, so safe nonviral systemic delivery methods for neonates are needed.

Hypothesis: If PEI-complexed plasmid DNA is injected intravenously into neonatal mice via the superior temporal vein, then effective systemic gene transfer to multiple organs can be achieved. A liver-specific AAT promoter/S/MAR plasmid should give liver-specific expression, but expression may be transient because of hepatocyte proliferation and loss of episomal vector genomes.

Aims: Develop a simple systemic intravenous injection method for PEI/DNA polyplexes in 1.5-day-old mice. - Optimize N/P ratio and DNA dose for transfection efficiency and neonatal survival. - Evaluate toxicity, inflammatory cytokine induction, and hepatic enzyme levels. - Assess biodistribution, tissue-specific expression, duration of expression, and vector genome persistence.

Delivery system: Platform: Nonviral PEI/DNA polyplexes for systemic neonatal gene transfer. - Polymer: Linear 22 kDa PEI (in vivo-JetPEI). - Payload: Plasmid DNA encoding luciferase: pUbC-Luc-S/MAR (ubiquitous human UbC promoter) and pLuca1 (liver-specific human alpha-1-antitrypsin/AAT promoter), both containing a Scaffold Matrix Attachment Region (S/MAR). - Formulation: PEI/DNA at N/P 8, complexed in 5% isotonic glucose, 30 µL total, injected via the superior temporal vein. - Targeting ligand: None; systemic delivery.

Approach: In vivo neonatal model: MF1 mouse pups injected at 1.5 days of age via the superior temporal vein. - Optimization: N/P ratios 4, 8, and 10; DNA doses 5 and 10 µg. N/P 8 with 10 µg selected for best expression and 93% survival (41/44). - Controls: Naked pDNA, 5% glucose negative control, LPS positive control for cytokine studies. - Time course: Bioluminescent imaging up to 25 days; tissues harvested at 24 h and later time points. No in vitro cell line experiments.

Key methods: Photon correlation spectroscopy for polyplex size. - In vivo bioluminescent imaging (IVIS) for luciferase expression. - ELISA for serum TNF-α, IL-12, and IFN-γ. - Hepatic enzyme assays for ALT, AST, and ALP. - Southern blot, methylation-dependent restriction assay, and quantitative PCR for vector genomes. - PCR for pDNA biodistribution in liver, lung, heart, spleen, brain, and kidney. - H&E histology and luciferase immunohistochemistry.

Key results: Polyplex size: ~143 ± 11 nm. - Optimization: Naked pDNA gave no detectable expression at 24 h. N/P 10 caused mortality (17/21 surviving), N/P 4 gave low expression, and N/P 8 gave strongest expression with 93% survival. 10 µg DNA gave ~10-fold higher expression than 5 µg at day 5 and ~20-fold higher at day 10. - Toxicity/inflammation: Transient cytokine induction: TNF-α ~400 pg/mL at 1 h for PEI/10 µg, undetectable by 18 h; IL-12 ~650 pg/mL at 6 h; IFN-γ ~6000–6500 pg/mL at 6 h, decreasing by 18 h. All lower than LPS. No inflammatory infiltrates on histology. No elevation of ALT, AST, or ALP at days 1, 5, or 10. - Biodistribution: pDNA detected in liver, lung, heart, spleen, brain, and kidney. Liver showed highest luciferase expression; lower expression in other tissues. - Liver-specific expression: pLuca1 expression was low at 24 h, dropped at day 3, rose to peak at days 11–12, then declined to background by day 20–25. By day 18 expression was 22.6% of peak, and 0.01% by day 24. Vector copies per 10 cells were ~4 at day 3, ~2.5 at day 5, ~4 at day 10, and ~0.5 at day 18; vector genomes were undetectable by day 25. No evidence of efficient plasmid replication by methylation-dependent restriction assay. - Safety: No developmental or behavioral abnormalities observed.

Interpretation: The authors conclude that systemic PEI/DNA delivery via the superior temporal vein is a simple, effective, and safe method for neonatal nonviral gene transfer. It produces broad tissue transfection and liver-specific expression that persists through the first two liver doublings, though expression is transient due to hepatocyte proliferation and episomal vector loss. This technique may enable early therapeutic applications requiring short-term gene expression and opens opportunities for nonviral neonatal gene therapy.

Limitations: Transgene expression was transient and declined to background by day 20–25. - S/MAR did not support detectable episomal plasmid replication in this neonatal model. - Only luciferase reporter genes were used; no disease model or therapeutic efficacy was tested. - Study was limited to neonatal mice; no larger animal validation. - Possible immune response to CpG motifs in pDNA was not fully resolved. - Injection technique is technically demanding due to small neonatal size. - No direct comparison with viral vectors or other nonviral formulations.