(419c) Searching for Optimal Properties of Synthetic Gene Vectors Via Simulations of Intracellular Transport Processes

Polymer-based nanocarriers for gene delivery vectors are being used extensively for in- vitro transfections and have also been used in clinical trials. However, they suffer from low efficiency which stems from the numerous barriers which retain or destroy a majority of the gene dose before it can reach the host nucleus. Thousands of different polymers have been used to enhance delivery efficiency, but only a few of these have been found to be significantly better than polyethylenimine (PEI^25kDa), the accepted standard in polymer-based gene delivery. Further, it is not clear whether these polymers, many of which have been developed and optimized for use with cultured cells, will be effective in in vivo applications. The most significant issue, however, is that even the best polyplexes are 1000-fold less efficient than viral vectors such as adenoviruses.

This remarkable difference between the best synthetic vector and a typical viral vector leads to a key question: Is there an inherent limitation to polyplexes? Or have we not found the magic polymer yet? In order to address this question we develop a detailed model of the gene delivery pathway which includes information about both the host cell and the vector, from the whole cell level to the single particle level. Firstly, the model shows that there exists a strong effect of cell morphology on the optimum intracellular itinerary of the vector. For flat, adherent, 2D cells in vitro the optimal pathway corresponds to enhancing the rates of all the steps involved. However, this is entirely due to the large number of vectors that enter directly on top the cell nucleus and are thus proximal to it. On the other hand, for ellipsoidal 3D cells in vivo the optimal pathway is much more complicated. There exist strong optima in the rates of endosomal escape and vector unpacking and greater enhancement in these rates actually reduces the delivery efficiency by up to 1000-fold. The optima themselves depend upon other parameters, showing that a systems analysis is essential to arrive at the optimal pathway.

On the absolute scale, the model predicts that the maximum delivery efficiency achievable by polyplexes even after optimization, is still very low compared to adenoviruses. This limit exists because of the low mobility of the vector in cell cytoplasm and its failure to protect DNA until the point of nuclear entry. New strategies for improved efficiency are proposed based on the findings from this study.