Intracellular barriers

Several major barriers need to be overcome for the development of nonviral gene delivery systems into true therapeutic products for use in humans. These barriers fall into three classes manufacturing, formulation, and stability (extracellular barriers and intracellular barriers) (85). Cationic lipids and cationic polymers self-assemble with DNA to form small particles that are suitable for cellular uptake. At the therapeutic doses positively charged particles readily aggregate as their concentration increases, and are quickly precipitated above their critical flocculation concentration. 

Lechardeur, D., and G.L. Lukacs. 2002. Intracellular barriers to non-viral gene transfer. Curr. Gene Ther. 2 183-194. 

Figure 6 Intracellular barriers to gene delivery include cellular uptake, intracellular transport, endosome escape, vector unpacking, and nuclear uptake. The gene carrier illustrated here has targeting moieties on the vector surface that are specific for cell surface receptors. The dotted arrow represents the prerequisite step of bypassing physiological barriers of the lung, such as the mucosal layer, and reaching the target cell surface.

The need to overcome intracellular barriers may require improved methods of determining rate-limiting steps for specific gene carriers, which will lead to the rational design of new carriers. To this end, our lab uses multiple-particle tracking (MPT) to investigate quantitatively the motion of nanometersized DNA delivery vehicles [170],... 

Steps taken to understand the intracellular barriers to gene delivery have led to the rational modification and improvement of carriers. Quantitative methods, such as multiple-particle tracking, to assess to the intracellular transport of gene carriers promise to add valuable insight that may ultimately lead to nonviral carriers rivaling the efficiencies seen in viral systems. 

Actively transported PEI/DNA nanocomplexes exhibited an average velocity of 0.2 pm/sec [118], a value on the same order of magnitude as motor-protein driven motion. Transport was revealed to be microtubule dependent, because both active transport and perinuclear accumulation were abolished upon microtubule depolymerization. Experiments utilizing MPT to quantify the other intracellular barriers to gene delivery are under way. 

Kitson C, Angel B, Judd D, Rothery S, Severs NJ, Dewar A, Huang L, Wadsworth SC, Cheng SH, Geddes DM, Alton EW. The extra- and intracellular barriers to lipid and adenovims-mediated pulmonary gene transfer in native sheep airway epithelium. Gene Ther 1999, 6, 534-546. 

To efficiently improve gene vectors, the intracellular barrier as the rate-limiting process for gene expression must be known. Furthermore, after modification of the devices, the issue of whether the modified... 

Genes can be delivered using either ex vivo or in vivo strategy. The ex vivo strategy encounters intracellular barriers presented by the cell that include the cell, endosome, and nuclear membranes (Fig. 6.38). 

Gene delivery to MSCs requires that DNA overcomes several intracellular barriers that can limit efficiency, including limited cellular internalization, endosomal escape, vector unpacking, and transport into the nucleus. 

For successful gene delivery, nonviral vectors have to be overcome at a number of extracellular as well as intracellular barriers until the carried DNA reaches its final destination, the nucleus. One strategy for overcoming the extracellular barriers in nonviral gene delivery is receptor-mediated endocytosis to enhance cellular uptake specifically. 

There are several steps in gene transfection such as the DNA complexation, cellular uptake of the complexes, release of DNA or complexes from endosomes, release of DNA from the carriers, and transfer into the nucleus (Zabner et al. 1995). Among them, an inefficient release of DNA in the polymer/DNA complexes from endosomes into the cytoplasm is one of the primary causes of poor transfection efficiency as the intracellular barrier. Therefore, pH-sensitive chitosan carriers have been tried to overcome the intracellular barrier. 

A major effort in the field of gene delivery is the development and evaluation of synthetic vectors to overcome the extra- and intracellular barriers encountered in the gene transfection pathway. The major barriers include... 

Once the cell has been targeted, the vector particle has to be internahzed by the cellular uptake machinery via endocytotic or phagocytotic uptake processes. This step can be taken rather easily by polyplexes, as appropriate receptor-binding ligands and/or cationic charges may enhance intracellular uptake of particles into endosomal vesicles. Several intracellular barriers then have to be overcome for successful transgene expression. Endosomal release was found to be a major bottleneck for many non-viral vectors [151,167]. The vector particle needs to survive and escape from the endosomal vesicular compartment, traffick the cytoplasmic environment, target the nucleus, enter the nucleus, and expose the carried nucleic acid to the cellular transcription machinery. 

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