Nucleic Acid Delivery: The Missing Pieces of the Puzzle?

Abstract

Conspectus The ability of gene or RNA interference (RNAi) delivery to increase or decrease virtually any protein in a cell opens the path for cures to most diseases that afflict humans. However, their high molecular weight, anionic nature, and instability in the presence of enzymes, pose major obstacles to nucleic acid delivery and frustrates their use as human therapies. This Account describes current ideas on the mechanisms in non-viral nucleic acid delivery and how lipidic and polymeric carriers overcome some of the critical barriers to delivery. A multitude of polymeric and lipidic vectors have been developed over the last 20 years, only a small fraction of them have progressed into clinical trials. Given that none of these vectors has received FDA approval, indicates that the current vectors do not yet have suitable properties for effective in vivo nucleic acid delivery. Nucleic acid delivery is a multistep process and inefficiencies at any stage result in a dramatic decrease in gene delivery or gene silencing. Despite this, the majority of studies investigating synthetic vectors focus solely on optimization of endosomal escape. A small number of studies address how to improve uptake via targeted delivery. A smaller fraction examine the intracellular fate of the delivery systems and nucleic acid cargo. The internalization of genes into the cell nucleus remains an inefficient and mysterious process. In the case of DNA delivery, strategies to increase and accelerate the migration of DNA through the cytoplasm and transport it through the nuclear membrane are required. The barriers to siRNA delivery are fewer: siRNA is more readily released from the carrier, siRNA is more resistant to enzymatic degradation and the target is in the cytoplasm; hence, siRNA delivery systems are becoming a clinical reality. With regard to siRNA therapy, the exact cytoplasmic location of RISC formation and activity is unknown. This makes specific targeting of the RISC for more efficient siRNA delivery difficult. Furthermore, identifying the factors favoring the binding of siRNA to Ago-2 and understanding how the half-life of siRNA and Ago-2/siRNA complex in the cytoplasm can be modulated without interfering with RISC functions that are essential for normal cell activity could increase siRNA delivery efficiency. In this manuscript we concisely review the current synthetic vectors and for a few of these, propose alternative strategies. We suggest how certain cellular mechanisms might be exploited to improve gene transfection and silencing. Finally, we raise the question if some carriers are delivering the siRNA to cells capable of repackaging the siRNA into exosomes. The exosomes would then transport the siRNA into a subsequent population of cells where the siRNA effect is manifest. This piggy-back mechanism may be responsible for reported deep tissue siRNA effects using certain carriers.

Chemistry specificity of DNA–polycation complex salt response: a simulation study of DNA, polylysine and polyethyleneimine

“…Therefore, it is reasonable that the proton sponge effect can cause endosomal rupture. However, in contrast to a common depiction of the proton sponge mechanism, 29,30,72 protonation and enchanced repulsion between protonated groups does not lead to swelling or loosening of the complex structure itself. Instead, our simulations show increased protonation leads to more stable and compact complexes.…”

Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery

“…S6) must escape this compartment (barrier 4, Fig. 1A) to gain access to the RNAi machinery (35,36). The relative capacity for LNP-mediated endosomal escape was simulated using a RBC hemolysis assay.…”

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

“…In contrast to DNA delivery, the destination of mRNA is the cytoplasm rather than the nucleus, and more genetic information can reach the target sites quicker due to reduced barriers during transfection because the inefficient nuclear internalization of transgenes is eliminated, and a shorter transport in cytoplasm will be needed [83]. mRNA can be translated almost instantly while DNA needs an additional transcription step.…”