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.

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.

Development of Lentiviral Vectors for HIV-1 Gene Therapy with Vif-Resistant APOBEC3G.

Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of restriction factor cytidine deaminase APOBEC3G (A3G), we developed self-activating lentiviral vectors that efficiently deliver HIV-1 Vif-resistant mutant A3G-D128K to target cells. To circumvent APOBEC3 expression in virus-producing cells, which diminishes virus infectivity, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, retroviral recombination between the direct repeats reconstituted an active A3G-D128K in 89%-98% of transduced cells. Lentiviral vectors that expressed A3G-D128K transduced CD34+ hematopoietic stem and progenitor cells with a high efficiency (>30%). A3G-D128K expression in T cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of several HIV-1 subtypes by C-to-U deamination leading to lethal G-to-A hypermutation and inhibition of reverse transcription. SIVmac239 and HIV-2 were not inhibited, since their Vifs degraded A3G-D128K. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for >3.5 months without detectable resistant virus, suggesting a high genetic barrier for the emergence of A3G-D128K resistance. Because of this, A3G-D128K expression in HIV-1 target cells is a potential anti-HIV gene therapy approach that could be combined with other therapies for the treatment and functional cure of HIV-1 infection.