Gary S. Koe

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.

Physical stability of nonviral plasmid-based therapeutics.

Nonviral, plasmid-based therapeutics are a new class of pharmaceutical agents that offer the potential to cure many diseases that are currently considered untreatable. While nonviral vectors have shown promise in clinical trials, their physical instability in liquid formulations represents a major barrier to the development of these agents as marketable products. While several different approaches have been used to improve the stability of liquid formulations, it is unclear whether aqueous suspensions can be rendered sufficiently stable to withstand the stresses associated with shipping and storage. Some studies have demonstrated the potential of frozen formulations to be stored for prolonged periods of time, however the potential for phase changes in frozen samples combined with the expense of maintaining the frozen state during shipping has stimulated an interest in developing dehydrated preparations. Although the stresses associated with dehydration are considerable, several studies have reported that sugars are capable of preserving the physical characteristics and transfection activity of nonviral vectors during acute lyophilization stress. This paper discusses the merits and drawbacks of the different approaches to preserving nonviral vectors, and identifies research areas in which more work is needed to develop stable formulations of plasmid-based therapeutics.

Infrared Spectroscopic Characterization of the Interaction of Cationic Lipids with Plasmid DNA

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and ζ potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.

Formulation Considerations for DNA-Based Therapeutics

The ability to deliver genes to cells and tissues in vivo offers the potential to develop potent vaccines and treat many hereditary diseases that are currently considered incurable, e.g., cancer, cystic fibrosis (CF), severe combined immunodeficiency (SCID), and acquired immune deficiency syndrome (AIDS) (1-6). Considering the tremendous promise of DNA-delivery technology, in addition to the extensive genetic information now available from the Human Genome Project, it is not surprising that gene therapy is being touted as the next revolution in medicine. Although a strict definition of “gene therapy” would be limited to therapeutic approaches that aim to use polynucleotides as a template for the in vivo production of proteins, the term is often used to refer to a wide variety of strategies that employ nucleotide-based molecules (e.g., vaccines, antisense, ribozymes, siRNA). To date, 70 clinical protocols have been approved for the delivery of naked DNA, comprising approx 11% of the total number of gene-therapy clinical protocols (http://www.wiley.co.uk/genetherapy/clinical). Slightly more studies (~12%) have employed nonviral, lipid-based vectors to facilitate DNA delivery. In comparison, the large majority of clinical gene therapy trials (>70%) utilizes viruses to deliver therapeutic genes because viruses are more efficient than contemporary synthetic gene-delivery systems. The higher efficiency of viruses should not be surprising if we recognize that these organisms have been evolving their gene-delivery machinery for billions of years. In contrast, the development of nonviral systems for therapeutic gene delivery can be traced back a mere 15 years (7). Although more efficient nonviral gene-delivery systems continue to be developed, synthetic systems have yet to replicate the efficiency of viruses. One significant drawback of viral delivery is the immunogenicity of viruses, which causes significant inflammation in vivo (8), and eliminates the potential for multiple dosing (anyone who has ever had a cold is familiar with the fever and inflammation associated with an immune response to viruses). In fact, the adverse reactions associated with viral delivery have been implicated as the cause of death in clinical trials (9,10). Also, a clinical trial involving liver infusion of an adenoassociated virus (AAV) for the treatment of hemophilia B was halted because of the presence of the viral vector in patient semen, and the concern that the genetic alteration could be passed to offspring (11). More recently, two patients treated with ex vivo genetherapy for the treatment of SCID have developed leukemia owing to insertional mutagenesis caused by the retroviral vector used in the study (12,13). Considering the potential safety risk involved in employing viruses as a therapeutic moiety, there is renewed interest in developing safe, efficient, nonviral gene-delivery systems.