Autophagy determines efficiency of liver‐directed gene therapy with adeno‐associated viral vectors

Abstract

Use of adeno‐associated viral (AAV) vectors for liver‐directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)–dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV‐mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short‐term treatment with a single dose of torin 1 significantly increased vector‐mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid‐α‐glucosidase in nonhuman primates. Conclusion: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver‐directed gene therapy using AAV vectors and showed in a proof‐of‐principle study how this virus–host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252–265).

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

“…3 Here, we demonstrated that exosome-enveloped AAV vectors offer a feasible, relatively simple strategy to achieve superior correction of hemophilia via enhanced targeting of hepatocytes, at the same time providing protection from preexisting NAbs. Although the mechanism of enhancement of transduction is not completely understood, based on our in vitro results, exo-AAV vectors seem to present a faster nuclear translocation rate, which may be the result of more efficient and autophagy-independent 22 intracellular trafficking. Additionally, competition experiments presented here suggest that exo-AAV entry occurs through a process dependent on the exosome membrane or protein components (eg, micropinocytosis, receptor-mediated endocytosis), which bypass the need of cell receptor binding of the AAV capsid.…”

“…Additional in vitro studies showed that exo-AAV, unlike their standard counterpart, do not seem to rely on autophagy to transduce hepatocytes, 22 as levels of LC3-II were unchanged in cells treated with exo-AAV vectors (supplemental Figure 2A-B). Furthermore, receptor saturation experiments with excess empty capsids showed inhibition of transduction of standard but not exo-AAV vectors (supplemental Figure 2C).…”

Emerging Issues in AAV-Mediated In Vivo Gene Therapy

“…Recently, autophagy activation through pharmacological inducers, such as clinically approved rapamycin, has been proven to increase hepatocyte transduction in small- and large-animal models; conversely, its inhibition resulted in decreased levels of AAV transduction. 123 …”

Host cell cytosolic immune response during Plasmodium liver stage development

“…Since adenoviruses are known to stimulate and exploit the host cell canonical autophagy machinery for their propagation (Hösel et al. 2017 ), limited resources might again be the reason for the increased parasite load, similar to the effect observed upon starvation. It will now be interesting to decipher the molecular details of this complex interplay between distinct autophagy pathways during starvation and virus infection that lead to the increase in parasite survival.…”