Caged DNA Does Not Aggregate in High Ionic Strength Solutions

Trubetskoy, Vladimir S.; Loomis, A. G.; Slattum, Paul M.; Hagstrom, James E.; Budker, Vladimir G.; Wolff, Jon A.

doi:10.1021/bc9801530

Cited by 87 publications

(67 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The authors, however, concluded that the crosslinked complexes lost their transfection activity and proposed therefore that it was necessary to use less stable disulfide linkage; notably the study included no microinjection component, so intracytoplasmic reduction could not be assessed directly. 6 In summary, this study shows that the apparently contradictory demands for successful systemic administration and efficient gene expression of nonviral gene delivery vectors can be addressed by design of complexes for activation by appropriate trigger mechanisms. Application of reduction-cleavable surface crosslinking provides extracellular stability, with extended plasma circulation times, coupled with efficient transgene expression following entry into target cells.…”

Section: Discussionmentioning

confidence: 89%

“…Recently, it was reported that crosslinked PLL/DNA complexes were less prone to saltinduced aggregation. 6 We therefore examined aggregation of PLL/DNA complexes in 0.15 m NaCl, but no significant effect of crosslinking was observed and both the crosslinked and non-crosslinked complexes aggre-gated extensively over 30 min, reaching sizes of ෂ1 m (data not shown). Fortunately, methods are available to increase the solubility of such PLL/DNA complexes, including surface modification of the complexes with PEG or other hydrophilic polymers.…”

Section: Figure 4 Transmission Electron Microscopy Of Dna Complexes mentioning

confidence: 99%

See 1 more Smart Citation

Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo

2001

View full text Add to dashboard Cite

We have developed polyelectrolyte gene delivery vectors that display good extracellular stability and are activated intracellularly to permit transgene expression. The strategy comprises covalent crosslinking of primary amines in poly-L-lysine/DNA complexes with a crosslinking agent that can later be cleaved by reduction. Crosslinked complexes maintained the same size and surface charge but showed increased stability against polyelectrolyte exchange with poly-L-aspartic acid. Surface modification with polyethyleneglycol improved solubility and masked their positive surface charge. Crosslinked complexes showed 10-fold increased plasma circulation following intravenous administration to Balb/c mice. In the absence of chloroquine, the levels of transgene expression in B16F10 murine melanoma cells were similar for crosslinked and non-crosslinked complexes, however, chloroquine selectively potentiated trans-

show abstract

Section: Discussionmentioning

confidence: 89%

Section: Figure 4 Transmission Electron Microscopy Of Dna Complexes mentioning

confidence: 99%

Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo

2001

View full text Add to dashboard Cite

show abstract

“…However, the slow reversal of the Schiff bases resulted in limited in vitro and in vivo gene expression. Other reversible cross-linking agents have also been applied to form caged DNA condensates (20) by template polymerization (21), but thus far they have not been shown to be transfection competent.…”

mentioning

confidence: 99%

A Potent New Class of Reductively Activated Peptide Gene Delivery Agents

McKenzie

Kwok

Rice

2000

Journal of Biological Chemistry

243

212

View full text Add to dashboard Cite

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-TrpLys 18 resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.A variety of nonviral gene delivery carriers have been developed and tested as in vitro transfection agents used to transiently express foreign DNA. Cationic lipids (1, 2), polylysine peptides (3-5), and cationic polymers such as polyethylenimine (6, 7) bind electrostatically to the phosphate backbone of DNA forming complexes that mediate cellular uptake of DNA in culture.As opposed to the success of these agents in vitro, attempted in vivo use has revealed many complications related to their toxicity (8), antigenicity (9), complement activation (10), solubility (11), blood compatibility (12), and stability (13). These complications relate to the size and charge of DNA carrier complexes and ultimately to the molecular characteristics of the carrier itself. High molecular weight (HMW) 1 DNA carriers can be cytotoxic (8), are able to activate the complement system (10), and can elicit an immune response (9). The size and heterogeneity of these polymers also significantly complicates region-specific derivatization with ligands or polyethylene glycol (14).To circumvent these problems, several low molecular weight (LMW) carrier peptides have been developed that mediate in vitro gene transfer as efficiently as their HMW counterparts (15-17). They offer the advantage of controlled synthesis and defined purity that then allows strategic optimization to increase expression levels and eliminate side effects.However, when analyzed for in vivo efficacy, LMW peptide DNA condensates lacked sufficient stability to survive circulation, were not able to significantly protect DNA from metabolism, and could not effect targeting (13,18). One solution to increase LMW pept...

show abstract

“…15,30,31,34 The ability to control the metabolic half-life of DNA co-condensates by changing the glutaraldehyde cross-linking level is a unique feature of this DNA formulation. The mechanism by which glutaraldehyde cross-linking delays DNA metabolism is most likely related to either caging of the DNA to protect it from DNase 35 or chemical masking of the peptide backbone of PEG-CWK 18 and Man9-CWK 18 from proteases by derivatization of the lysine side chains. 23 The results presented in Figure 4 indicate that increasing the glutaraldehyde cross-linking from 6 to 15 equiv nearly doubles the t 1/2 from 3.7 to 6 h and similarly in¯uences the level of transient gene expression ( Figures 6A, versus 6C).…”

Section: Discussionmentioning

confidence: 99%

Cross‐linked low molecular weight glycopeptide‐mediated gene delivery: Relationship between DNA metabolic stability and the level of transient gene expression in vivo

Yang

Park

Man

et al. 2001

Journal of Pharmaceutical Sciences

View full text Add to dashboard Cite

DNA co-condensates were formed by reacting [125 I]DNA with an admixture of a high-mannose glycopeptide (Man9-CWK 18 ) and either of two poly (ethylene glycol) peptides (PEG-VS-CWK 18 or PEG-SS-CWK 18 ) followed by crosslinking with 6±50 mol equiv of glutaraldehyde. [ 125 I]DNA co-condensates were administered intravenously in mice to determine the in¯uence of peptide DNA formulation parameters on speci®c targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9-CWK 18 and PEG-CWK 18 to mediate speci®c recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross-linked Man9-CWK 18 /PEG-CWK 18 DNA co-condensates was receptor mediated since Kupffer cell targeting was inhibited by pre-administration of Manbovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver-targeted DNA localized to Kupffer cells. Crosslinking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half-live-of 4, 6, and 39 h, respectively. Tail vein dosing of 50 mg of DNA co-condensates cross-linked with 6 mol equiv of glutaraldehyde produced detectable levels of human a 1 -antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human a 1 -antitrypsin was elevated two-fold each day when dosing with DNA co-condensates cross-linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co-condensates cross-linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half-life, providing insight into a means to target DNA condensates to peripheral tissues. ß

show abstract

Caged DNA Does Not Aggregate in High Ionic Strength Solutions

Cited by 87 publications

References 22 publications

Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo

Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo

A Potent New Class of Reductively Activated Peptide Gene Delivery Agents

Cross‐linked low molecular weight glycopeptide‐mediated gene delivery: Relationship between DNA metabolic stability and the level of transient gene expression in vivo

Contact Info

Product

Resources

About