Overcoming nonviral gene delivery barriers: perspective and future

Abstract

A key end goal of gene delivery research is to develop clinically relevant vectors that can be used to combat elusive diseases such as AIDS. Despite promising engineering strategies, efficiency and ultimately gene modulation efficacy of nonviral vectors have been hindered by numerous in vitro and in vivo barriers that have resulted in subviral performance. In this perspective, we concentrate on the gene delivery barriers associated with the two most common classes of nonviral vectors, cationic-based lipids and polymers. We present the existing delivery barriers and summarize current vector-specific strategies to overcome said barriers.

Figure 1

Chemical structures of common cationic lipids used in transfection.

Figure 2

Structural variants of the PEI cationic polymer.

Figure 3

Three basic structural domains of cationic lipids: hydrophobic tail, linker, and headgroup.

Figure 4

Schematic representation of the packing efficiency of cationic lipids and their resulting phase structure. A packing parameter, $P=\frac{v}{{aL}_c}$, less than ½ confers a cone-like shaped structure that assembles into micellar phases. Values between ½ and 1 allow formation of the lamellar phase (bilayers). For P > 1, the negative curvature leads to the formation of the inverted hexagonal phase.

Figure 5

Schematic representation of classical and proposed lipid mixing mechanisms. a) Classical lipid mixing mechanism was believed to proceed by membrane fusion of the lipoplex and host endosomal membranes followed by exchange of negatively charged phospholipids from the cytoplasmic to the inner face of the endosome. Membrane destabilization and nucleic acid release occur as a result. b) Conversely, lipid mixing may function by an altered mechanism where lipoplexes that are in close proximity to the inner-endosomal membrane lose lipid molecules through dispersal or degradation. Release of lipid molecules permits the passive release of nucleic acids, followed by subsequent cytoplasmic transport mediated by transient pore formation resulting from integration of freed lipids.

Figure 6

Schematic representation of classical and proposed proton-sponge mechanisms. a) Classical polyplex-induced proton sponge effect proceeds by the increase of protons due to protonation of amines in the polyplex and ATPase-mediated H⁺ influx. This is followed by concomitant influx of both counter-ions (Cl⁻) and water into endocytic vesicles, resulting in osmotic-induced lysis. However, recent studies support an alternative mechanism b) where nucleic acid release relies on a time-depend protonation-induced membrane permeabilization by tight binding of the polyplex and the inner endosomal membrane.