Identification of mechanisms that contribute to the resistance against lentiviral gene delivery in human NK cells.

Especially in cells of the innate immune system, the success of viral gene delivery has always proven challenging and less efficient than other cells of hematopoietic origin. The major hurdle in gene therapy is the delivery of the gene-of-interest into the cell. When it comes to hijacking cellular defense mechanisms and carrying genetic information into the cell, nature presents us with an evolutionarily perfected carrier: viruses. However, using viruses for gene delivery has problems of its own. In order to get rid of the pathogenicity of the virus and ensure that the virus will not be replicating and spreading, all viral genes and sequences except those necessary for packaging of the viral genome are removed during viral vector production.

As with wild-type viruses, intracellular recognition of viral components by pattern recognition receptors is a possible mechanism of cellular response against viral vectors. It is well known that toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) play a major role in the detection of internalized viral particles and induction of an anti-viral state. Many wild-type viruses have developed elaborate schemes to avoid detection by these receptors and increase their virulence. In the case of viral vectors, the removal of various viral genes that counteract host responses but are dispensable for vector production is often preferred due to safety and practicality considerations. Inevitably, this would render viral vectors more prone to inducing strong innate responses upon target cell infection. It is possible that TLR or RLR mediated detection of viral vector components might activate an anti-viral response, negatively affecting the efficiency of viral gene delivery.

In order to test this hypothesis, we have previously attempted to use small molecule inhibitors of TLR and RLR signaling preceding lentiviral transduction, and observed a dramatic increase in gene delivery efficiency. Therefore, it might be possible to state that the lentiviral RNA is recognized by one or more of these receptors and an anti-viral response is triggered.

These results present a proof-of-principle for the feasibility of such approaches for enhancement of gene therapy applications. Overcoming the intracellular defense mechanisms against gene delivery vectors will lead to a dramatically increased delivery rate of the transgene. This may have broad technical applications in order to improve the efficiency of genetic modification of a wide variety of cell types. Because of the innate resistance of NK cells against viral infections, compared to other cell types, they are a good model to get detailed insights about cellular response mechanisms against viral vectors.