Overcoming Gene-Delivery Hurdles: Physiological Considerations for Nonviral Vectors

Abstract

With the use of contemporary tools and techniques, it has become possible to more precisely tune the biochemical mechanisms associated with using nonviral vectors for gene delivery. Consequently, nonviral vectors can incorporate numerous vector compositions and types of genetic cargo to develop diverse genetic therapies. Despite these advantages, gene-delivery strategies using nonviral vectors have poorly translated into clinical success due to preclinical experimental design considerations that inadequately predict therapeutic efficacy. Furthermore, the manufacturing and distribution processes are critical considerations for clinical application that should be considered when developing therapeutic platforms. In this review, we evaluate potential avenues towards improving the transition of gene-delivery technologies from in vitro assessment to human clinical therapy.

Figure 1

General gene delivery mechanism. Upon assembly of the chosen nucleic acid cargo with the delivery vector construct, the composite particles must traverse various extracellular barrier (e.g., serum endonucleases) followed by gaining cellular entry through endocytosis (depicted in the figure) or by other means. Following uptake, particles modulate gene expression by either in the cytosol (expression-independent) or in the nucleus (expression-dependent). Regardless of the strategy selected, a gene delivery vector must successfully navigate in vitro and in vivo testing and GMP manufacturing prior to entering clinical testing.

Figure 2

Compositional design considerations for three representative properties of nonviral gene delivery vectors. Genetic cargo is surrounded by a coating (green outline) that provides protection and/or cell-specific targeting (top left). Furthermore, the vector itself can be designed from a variety of different chemical compositions (top right) and constructed into various shapes (bottom left). This flexibility in compositional design allows researchers to tune gene delivery vectors for specific clinical applications.

Figure 3

Physical design considerations of in vitro systems vs. in vivo conditions. Gene therapy studies typically incubate delivery transfection agents (red [A] and blue [B]) under conditions that are not representative of systemic in vivo circulation. One such condition is the use of long incubation times (A), which are physiologically unrealizable and can be avoided by using microelectromechanical systems (MEMS). Additionally, in vitro measurements of preferential cell uptake are usually validated using homogenous cell populations in media lacking relevant levels of potentially interfering serum proteins (green particles) (B). Transfection studies can be improved by determining delivery specificity metrics in physiological media with heterogeneous cell populations to accurately predict non-specific interactions with serum proteins and off-target delivery respectively.