Structural Alterations of Gene Complexes by Cystic Fibrosis Sputum

Abstract

Gene complexes with optimal physicochemical characteristics for cystic fibrosis (CF) gene therapy in vitro may become inactive in vivo as a result of destruction upon interaction with CF mucus. Therefore, we examined in this study to what extent main sputum components (linear DNA, mucin, phosphatidylcholine, phosphatidylglycerol, and albumin) may disintegrate lipoplexes. We found that mixing linear DNA with lipoplexes, in concentration ratios as occurs in the mucus of patients with CF in clinical studies with lipoplexes, drastically altered the surface charge and size of our lipoplexes and resulted in the liberation of plasmid DNA from the lipoplexes. These concentration ratios occur in vivo when the DNA concentration in the sputum becomes > 2.7 mg/ml, a quite realistic concentration even in patients without acute exacerbations. Lipoplexes brought in contact with native CF sputa at clinically relevant concentration ratios dissociated when the DNA concentration in the sputa was > 2.7 mg/ml. However, when the linear DNA was degraded by recombinant human deoxyribonuclease I before lipoplexes were added, the linear DNA did not cause any dissociation of the lipoplexes. Addition of albumin and mucin to the lipoplexes in a clinically relevant concentration ratio changed the surface charge of the lipoplexes to negative, however, without release of plasmid DNA. Mucin, dipalmitoylglycerophosphocholine, and dipalmitoylglycerophosphoglycerol did not cause any change in lipoplex properties at clinically relevant concentration ratios.

Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus

“…In a previous study we experimentally showed that lipoplexes mixed with CF sputa indeed started to dissociate when the linear DNA:pDNA concentration ratio in the mixtures was above 1.6. 8 From the dissociation experiments on the GL67 lipoplexes by CF mucus components we showed that only albumin was able to disassemble these lipoplexes (although artificially) at concentration ratios that exist in vivo (Tables 2 and 3). Indeed, dissociation was observed from an albumin:pDNA concentration ratio of 40 onwards.…”

“…Indeed, we recently showed that CF mucus components clearly interact physicochemically with cationic lipoplexes. 8 However, besides this report, there is no additional information pertaining to this matter. In particular, it has not been determined how structural changes to the gene complexes by CF mucus constituents influence their transfection activity.…”

“…8,22,23 Others have reported that Alveofact, a natural pulmonary surfactant, forms a barrier to cationic lipid-mediated gene transfer. [11][12][13] We examined whether this also held true for our lipoplexes.…”

“…The Na + , Cl -and Ca 2+ concentrations in the latter buffer represent the average concentration of these ions in CF sputum. 8 …”

“…CF mucus contains large amounts of proteins, DNA, mucin and phospholipids 8 and may decrease gene transfer to the underlying epithelium in different ways: (1) may sterically restrict the transport of the gene complexes to the target cells. We studied this transport barrier in our previous work.…”