Aerosol delivery of plasmid DNA to the lungs offers the possibility of direct application of gene preparations to pulmonary surfaces as a means of treating a variety of genetic pulmonary disorders. However, the process of jet nebulization rapidly degrades naked DNA, viral vectors, and many lipid-based formulations. While complexing DNA with cationic lipids has been shown to significantly stabilize plasmid DNA, losses of biological activity often occur during nebulization, severely limiting the efficiency of aerosol delivery of many such complexes. In conjunction with the design of aerosol delivery systems appropriate for DNA delivery, we have developed formulations using polyethyleneimine (PEI, a polycationic polymer) and DNA that result in a high level of pulmonary transfection (10- to 100-fold greater than many cationic lipids) and are stable during nebulization. In addition, these PEI-based formulations exhibit a high degree of specificity for the lungs. The properties of PEI-based formulations that make them resistant to nebulization and efficient as DNA delivery vectors for pulmonary sites have been investigated. Potential applications of this technology, including the use of aerosolized PEI-DNA for genetic immunization, are discussed.
Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i.v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25–100 μm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.
A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.
Attachment of 6,9-diamino-2-methoxyacridine to the 5' end of a purine-rich oligodeoxynucleotide targeting a 15 bp oligopurine oligopyrimidine stretch in the promoter region of the interleukin-2 receptor alpha chain (IL-2R alpha) gene results in an approximately 500-fold increase in its triplex forming avidity as determined by both band shift assay and DMS footprinting (Kd lowered from 2.5 microM to 5 nM). This oligonucleotide participates in Mg(2+)-dependent three-stranded DNA formation in which it is oriented antiparallel relative to the purine strand of the target duplex as determined by acridine moiety sensitized photoreactivity with the target duplex DNA. The oligonucleotides used in these studies were synthesized with a 3-amino-2-hydroxypropyl group at the 3' end to protect against exonucleolytic degradation for future in vivo applications. The 3'-amino group underwent partial removal, probably during the NaOH deprotection step. Both the 3'-amino and the 3'-free forms of the oligo have the same binding avidity and specificity. The interaction of the third strand with its target is sequence specific and can be essentially abolished by a point G-->T transversion 4 bases away from the 3' end of the target oligopurine block or severely reduced by other mutations within the target duplex. Thus, the attachment of the acridine moiety to the 5' end of the oligonucleotide does not seem to substantially compromise the sequence specificity of binding. Additionally, the oligonucleotide composed of G and A nucleotides was found to be superior to the oligonucleotide containing G and T residues since the difference in avidity of binding to the same target site was 17-fold.
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