Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets

Abstract

Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.

Keywords: gene therapy; lungs; newborn pigs; non-viral vector; tetrafunctional block copolymers.

Graphical abstract

Figure 1

Anatomical Representation of Pig Lungs The trachea and the different lobes of the lungs are represented.

Figure 2

Liposomal Gene Transfer to Newborn Pig Lungs CholP/DOPE/DMPE-PEG5000 liposome formulation was used to deliver 1 mg plasmid. Forty-eight hours later, the animals were euthanized, lungs were cut into small cubes, and CAT activity was measured in individual cubes. (A and B) Transgene expression in individual cubes and the repartition in the different lung compartments. The total amount of CAT protein measured in each lung compartment of pig n° 1404 (A) and pig n° 1412 (B) are presented in (C) and (D), respectively.

Figure 3

Tetrafunctional Block Copolymer-Mediated Gene Delivery of 0.5 mg Plasmid The 704-DNA formulation was used to deliver 0.5 mg plasmid. Forty-eight hours later, the animals were euthanized, lungs were cut into small cubes, and CAT activity was measured in individual cubes. (A) Transgene expression in individual cubes and the repartition in the different lung compartments. The total amount of CAT protein measured in each lung compartment of pig n° 1402 (A) is presented in (B).

Figure 4

Tetrafunctional Block Copolymer-Mediated Gene Delivery of 1 mg Plasmid The 704-DNA formulation was used to deliver 1 mg plasmid. Twenty-four hours (A and D) or forty-eight hours (B, C, E, and F) later, the animals were euthanized, lungs were cut in small cubes, and CAT activity was measured in individual cubes. (A–C) Transgene expression in individual cubes and the repartition in the different lung compartments. The total amount of CAT protein measured in each lung compartment of pig n° 1411 (A), pig n° 1403 (B), and pig n° 1410 (C) are presented in (D), (E), and (F), respectively.

Figure 5

Comparison of the Total Amount of CAT Protein Produced in the Whole Lung of Liposome- and Tetrafunctional Block Copolymer-Mediated Gene Delivery of 1 mg Plasmid The liposomal-DNA or 704-DNA formulation was used to deliver 1 mg plasmid. Forty-eight hours later, the animals were euthanized, lungs were cut into small cubes, and CAT activity was measured in individual cubes. CAT protein amounts in the whole lungs of newborn pigs were added. The data represent the mean ± SEM of n = 2.

Figure 6

IL-6 Expression in Response to Tetrafunctional Block Copolymer-Mediated Gene Delivery of 1 mg Plasmid Lung IL-6 contents were determined by enzyme-linked immunosorbent assay in the different lung compartments after intratracheal instillation of saline buffer (pig n° 1401) or 1 mg plasmid DNA complexed with the tetrafunctional block copolymer (pig n° 1403).