Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

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.

mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3.

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.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EµB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

An optimized measles virus glycoprotein-pseudotyped lentiviral vector production system to promote efficient transduction of human primary B cells.

Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%), but suffers from low titers. To overcome current limitations, we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction, including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency, and ddPCR for VCN analysis.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.

CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells.

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.