In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes

Purpose: First-generation cationic liposome–DNA complexes showed insufficient in vivo gene transfer efficiency. A more virus-like, self-assembling non-viral vector was needed that could condense and protect plasmid DNA and produce efficient gene expression after systemic administration.

Hypothesis: If plasmid DNA is first condensed with protamine sulfate and then coated with DOTAP cationic liposomes to form LPD complexes, then the DNA will be better protected from enzymatic degradation and will produce higher in vivo gene expression after intravenous injection than DOTAP/DNA complexes alone.

Aims: Develop and characterize LPD complexes composed of DOTAP, protamine, and plasmid DNA. - Evaluate in vivo gene expression after intravenous administration in mice, including charge-ratio, dose, time-course, and tissue-distribution effects. - Compare LPD with DOTAP/DNA and protamine/DNA complexes, and test different polymers and administration routes. - Identify transfected cell types using a lacZ reporter and assess preliminary toxicity.

Delivery system: Platform: Cationic lipid–protamine–DNA (LPD) complexes. - Components: Plasmid DNA; protamine sulfate; DOTAP cationic liposomes. - Formulation: Protamine mixed with DNA at a 1:1 (+/−) charge ratio, followed by DOTAP liposomes at high DOTAP/DNA ratios (e.g., 11:1, 23:1, 29:1, 35:1 nmol/µg). - Payload: pCMVL (firefly luciferase) or pCMVLacZ (β-galactosidase) reporter plasmids. - Targeting ligand: None. - Route: Intravenous tail-vein injection; intraportal injection tested for comparison. - Size: LPD complexes approximately 200–300 nm.

Approach: Model: Female CD-1 mice, 4–6 weeks old; groups of three mice unless otherwise noted. - Dose: Typically 50 µg DNA per mouse; dose escalation to 25, 50, 75, and 100 µg. - Controls: DOTAP/DNA complexes, protamine/DNA complexes, naked DNA, different polymers (polylysine, protamine-free base, protamine phosphate, protamine sulfate). - Readouts: Luciferase activity in heart, lung, liver, spleen, and kidney; tissue distribution of radiolabeled DNA/lipid; Southern blot; DNase I protection; X-gal staining; toxicity.

Key methods: Luciferase activity assay in tissue homogenates, normalized to protein. - Dynamic laser light scattering for particle size. - Radioactive labeling of DNA with ¹²⁵I and liposomes with ¹¹¹In for biodistribution. - Southern blot analysis of plasmid DNA extracted from tissues. - In vitro DNase I protection assay. - X-gal histochemical staining for lacZ expression. - Histological examination for inflammation.

Key results: LPD gave consistently higher in vivo gene expression than DOTAP/DNA complexes after intravenous injection. - Highest expression was in the lung; expression was also detected in heart, spleen, liver, and kidney. - Gene expression was charge-ratio dependent; higher DOTAP/DNA ratios increased expression, especially at low DNA doses. - Optimal dose was approximately 50 µg DNA per mouse; at this dose, about 20 ng luciferase per mg extracted tissue protein was detected in lung. - Increasing DNA to 100 µg per mouse caused toxicity and death. - Expression was detected as early as 1 h, peaked at 6 h, and declined thereafter; lung expression lasted up to 2 days, with detectable signal at 4 days in lung, spleen, and liver. - Intraportal injection led to about a 100-fold decrease in lung gene expression compared with intravenous injection. - Endothelial cells were the primary transfected cell type in lung and spleen; no obvious inflammation was observed. - LPD protected DNA from DNase I digestion better than DOTAP/DNA complexes.

Interpretation: The authors conclude that LPD is a novel, efficient non-viral vector for in vivo gene transfer. Because protamine sulfate is nontoxic and only weakly immunogenic in humans, LPD may be useful for clinical gene therapy, particularly for lung-targeted intravenous gene delivery.

Limitations: Only mouse studies; no large-animal or human validation. - High DNA doses (100 µg/mouse) caused toxicity and death. - Gene expression was transient, peaking at 6 h and declining rapidly. - Considerable variability in expression was noted, especially with DOTAP/DNA complexes and sequential injections. - No therapeutic efficacy endpoint; only reporter genes. - No long-term safety, immunogenicity, or repeated-dose evaluation. - No active targeting ligand; lung accumulation was largely passive/charge-dependent. - Old study; formulations and analytical methods are not state-of-the-art.