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Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV. H/F LVs produced in CD46 null HEK293T cells, generated by CRISPR/Cas9-mediated knockout of CD46, produced 2-fold higher titer vector compared with LVs produced in CD46+ HEK293T cells. This resulted in approximately 2- to 3-fold higher transduction of HSPCs while significantly reducing target cell cytotoxicity caused by producer cell contaminates. Improved H/F LV entry into HSPCs and distinct entry mechanisms compared with VSV-G LV were also observed by confocal microscopy. Given that vector production is a major source of cost and variability in clinical trials of gene therapy, we propose that the use of CD46 null packaging cells may help to address these challenges., Graphical Abstract

Significance Gene delivery by virus-like particles holds enormous therapeutic potential to correct inherited genetic disorders and to prevent infectious disease. However, cells express antiviral factors that prevent virus infection and, consequently, limit the success of gene therapy. Here, we reveal the mechanism by which the drug rapamycin improves lentivirus-mediated gene delivery. Rapamycin treatment led to degradation of IFITM3, a broad and potent antiviral protein which inhibits virus entry into cells. IFITM3 is selectively cleared from endosomes, the sites where viral and cellular membranes fuse, and is sorted for disposal in lysosomes. While revealing an immunosuppressive function with clinical benefits, we caution that rapamycin use in humans may facilitate infection by pathogenic viruses like Influenza A virus., Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are well-established immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus–cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

A major obstacle to the success of gene therapy for hematologic disorders is the inefficiency of lentiviral vector (LV) gene transfer to hematopoietic stem cells (HSCs). Achieving clinically relevant gene delivery levels requires prolonged ex vivo culture of HSCs with cytokines and large LV doses. Rapamycin, PGE2, and other small molecules, have been reported to increase LV transduction of HSCs, however the mechanism of action of these drugs and the basis for LV restriction in HSCs are poorly understood.Here, we report a novel small molecule, β-deliverin, which improves LV gene transfer to HSCs up to 3 fold over DMSO control in vitro. This effect is most pronounced in human adult peripheral blood-derived HSCs, but also observed in human cord-derived HSCs, and in non-human primate bone marrow-derived HSCs. Importantly, treatment with β-deliverin does not significantly affect the viability or expansion of HSCs in culture. Furthermore, for cord-derived HSCs, there was no reduction in the number or type of colony-forming cells. In vivo, β-deliverin treated HSCs engraft in the peripheral blood of NSG mice at levels comparable to control at 16 weeks post-engraftment. Despite an only modest increase in transduction efficiency in β-deliverin-treated cord-derived HSCs transduced in vitro (26% versus 20% for control), the marking frequency for control cells dropped to