Unexpected help: mTOR meets lentiviral vectors

In this issue of Blood, Wang et al show that rapamycin facilitates lentiviral transduction in human and mouse hematopoietic stem cells (HSCs) both in vitro and in vivo via increased cytoplasmic release of vector particles.¹ 

Since the earliest successes at gene transfer into HSCs,²  the efficiency of transfer into repopulating HSCs has been a barrier that has limited applications to human therapeutic approaches. Optimization of gene transfer vectors, vector production, ex vivo culture, and transduction conditions as well as conditioning regimens have paved the way for the success of a number of recent gene therapy trials. In some of these trials, long-term engraftment and clinical benefit was observed even in the absence of an in vivo selective advantage of gene-modified cells.³  Despite these advances, there is room for improvement to further enhance the outcome and reduce the costs per patient. For example, genetic modification of HSCs using lentivirus vectors requires high vector doses due to cell-intrinsic barriers hampering retroviral transduction. Increasing the efficiency of this process is not only of interest for laboratory researchers but is also particularly relevant for clinical gene therapy trials, where the efficacy of some therapies and the total number of patients that can be treated with a given lot of certified vector, produced via costly Good Manufacturing Processes, directly depends on the efficiency of HSC transduction. One step into this direction is described in the current article by Wang et al, where the authors show about a fourfold enhanced transduction of HSCs in the presence of rapamycin. This approach adds to previously published transduction enhancers such as polybrene, fibronectin, semen-derived enhancer of virus infection (SEVI), and protamine sulfate.

Wang et al also set out to elucidate the cellular mechanisms underlying the rapamycin-mediated increase in transduction. Rapamycin inhibits mammalian target of rapamycin (mTOR) by interfering with the binding of essential coactivator proteins, thereby influencing a number of different physiological functions, such as protein translation and autophagy.⁴  Wang et al could clearly show that inhibition of mTOR is essential in mediating the rapamycin effect, but autophagy does not play a role. This observation is not too surprising, given the fact that previously established links between autophagy and HIV-1 lifecycle are limited to postintegration events, such as maturation of the gag-precursor protein and virus shedding, both irrelevant for transduction. Other known modulators of retrovirus transduction also did not seem to be influenced by rapamycin treatment, including Trim5, CypA, or p21.⁵  The critical step appears to occur after uptake of viral particles but before the preintegration complex is formed, identifying the endosome escape/cytoplasmatic entry as the crucial events (see figure). The essential cellular components that are modulated by mTOR inhibition to mediate this effect still remain to be identified but could also provide insight into so-far-undescribed functions of mTOR signaling in HSCs.

Importantly, rapamycin might easily be adapted to the clinical setting, as it is currently being tested in a variety of clinical trials and is used in patients to prevent organ transplant rejection and to treat certain type of cancers. In contrast to polybrene, fibronectin, protamine sulfate, and SEVI, which increase nonspecific binding of vector particles to the cell surface,⁶  as noted above rapamycin acts on an early postentry step of viral infection. Other cell culture additives (apart from cytokines) that modulate cellular processes to aid viral transduction are the proteasomal inhibitor MG132 and, as very recently published, prostaglandin E2.⁷  In both cases, the relevant cellular mechanisms remain largely unknown. Application of some of these transduction enhancers has a negative impact on cell physiology and vitality, although rapamycin treatment appears to have a useful side effect: HSCs are pushed into a quiescent state. The immediate consequence is that reduced cell numbers are available for transplantation, but this is fully compensated for by apparently superior engraftment of rapamycin-treated cells, thereby even potentially amplifying the effect of enhanced transduction in the transplantation setting. Thus, in addition to improved transduction rates, this procedure is a step closer toward the “holy grail” of ex vivo culture of HSCs in a gene transfer setting, that is, to maintain the “stemness” of cells over several days in culture.⁸  In this regard, rapamycin is similar to effects seen on HSC transduced in the presence of fibronection (Retronectin).⁹  Rapamycin also apparently facilitates in vivo transduction, and is the first agent to display these properties. This is relevant as the degree of HSC gene marking is particularly low in this type of application.

It is not clear whether rapamycin administration is helpful for other promising vector platforms used for in vivo transduction, for example, adeno-associated virus and retargeted retroviral vectors pseudotyped with modified measles virus envelope,¹⁰  as they seem to use clathrin-independent entry pathways. Furthermore, future studies have to prove the effectiveness of rapamycin-assisted transduction using granulocyte colony-stimulating factor–mobilized peripheral blood CD34⁺ cells, the relevant target population in many gene therapy protocols, which are notably difficult to transduce. Finally, given the mode of action of rapamycin, it will be critical that use of this agent in large-scale preclinical testing and clinical trials is shown not to affect human stem cell engraftment in a negative fashion. Confirmatory testing in a broad spectrum of applications by other laboratories will be critical to future broad acceptance of this exciting new approach to HSC gene transfer.