Biological barriers to cellular delivery of lipid-based DNA carriers

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Abstract

Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure/activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.

Section snippets

Foreword

The challenge that one faces when writing a review is to identify a focus. This is a difficult task in the area of gene therapy because the technologies and therapeutic applications are so diverse. A brief search of the MEDLINE data base of scientific literature indicated that over 300 reviews on gene therapy were published over a 9 month period ending in September 1997, the time when this review was prepared. A cursory examination of these reviews leaves one with the feeling that researchers

Introduction to the technology

Recent advances in molecular genetics have contributed significantly to our understanding of disease pathogenesis [1], [2]. A logical extension of this new understanding concerns the development of therapeutic strategies that specifically target genetic abnormalities [3], [4]. Advances in molecular biology have resulted in a new concept for treatment of disease, the concept of gene therapy. Gene therapy can be defined as any treatment strategy that relies on the introduction of polynucleotides

Defining the ‘activities’ of lipid-based plasmid delivery systems

In order to develop useful plasmid delivery systems, the carrier formulation must fulfill many ‘activities’ in order to facilitate transgene expression. If the activity of the delivery system is defined in a narrow context, then one can judiciously address how individual components of the delivery systems affect specific requirements, such as protection of the associated DNA. Although six steps have been defined in Fig. 1 as requirements for a therapeutically useful gene transfer system, in the

Preparation of lipid–DNA complexes

The concept of using lipid-based carriers for delivery of DNA to cells resulted from an extensive amount of research on the use of liposomes as drug carriers [40]. As DNA carriers, this technology relied on capturing DNA within the aqueous compartments of multilamellar liposomes during their formation; a simple process requiring hydration of dried lipids with a solution containing DNA. The liposomes were typically prepared using neutral and/or anionic lipids and under ideal conditions these

The first barrier: protection of the plasmid expression vector

The physical characteristics of the lipoplexes being developed for delivery of plasmid expression vectors must protect the associated DNA from factors that will prevent transgene expression. Superficially, lipid-mediated protection of plasmid expression vectors is easy to measure, easy to achieve and most approaches used to prepare lipoplexes result in some level of DNA protection, as assessed by inhibition of DNA intercalating dye binding and DNA stability in the presence of DNase I or serum

The second barrier: binding to the cell membrane

In general, lipoplex formulations that give rise to optimal transfection in vitro and in vivo are prepared such that the structure generated exhibits a net positive charge [63], [64]. It has been argued that the positive charge facilitates binding to cell membranes, which have an anionic surface. We believe that this argument is too simplistic. As suggested above, lipoplex formulations tend to aggregate in the presence of salts and this aggregation reaction can be attributed to the surface

The third barrier: DNA delivery and dissociation from the carrier

Once a lipoplex interacts with a cell membrane, DNA enters the cell either directly through the plasma membrane or indirectly following endocytosis. Both entry routes require membrane destabilization and, regardless of whether the destabilized membrane is the plasma membrane or the endosomal membrane, these and/or other intracellular processes must also involve dissociation of the plasmid expression vector from the lipoplex. A pivotal manuscript addressing potential mechanism(s) of

The fourth barrier: transfer into the nucleus

Cationic lipid-mediated transfection is a relatively inefficient process, which is initiated by binding of the plasmid expression vector to the cell [64], [98]. While a significant portion of the cell-associated DNA is internalized, only a small percentage of DNA is released from endosomes. Therefore, only a small amount of DNA will be available as free DNA in the cytoplasm, and this DNA will still be susceptible to degradation. This free DNA must still find its way to the nucleus. An unlikely

Summary – lipoplex design challenges

Our interest in developing lipid-based carriers with attributes that change as a function of time following i.v. administration has been summarized here. Other investigators are focusing on developing model (reconstituted) viruses as efficient nucleic acid delivery systems. The three viral attributes that have attracted the greatest attention are (i) surface binding interactions [125], (ii) viral membrane fusion proteins [126] and (iii) nuclear targeting signals [127]. Based on what is known

Acknowledgements

The Medical Research Council of Canada and Inex Pharmaceuticals Corp. supports research on gene transfer formulations in M. Bally’s laboratory. D. Reimer was a MRC Postdoctoral Fellow. E. Wasan holds a fellowship from the British Columbia Science Council. F. Wong is a recipient of a Cancer Research Society Studentship.

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