b-globin lentiviral vectors (b-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the b-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a b-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the fulllength parental b-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a b-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

Summary Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3′ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR−/− cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.

X-Linked Chronic Granulomatous Disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene resulting in the inability of phagocytic cells to eliminate infections. In order to design a lentiviral vector capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was utilized to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600 kb topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated lentiviral vector (LV) for the treatment of X-CGD. When compared to a LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against B. cepacia infection, and restored healthy donor levels of anti-microbial oxidase activity in neutrophils derived from X-CGD patient HSPCs. Our findings validate the bioinformatics-guided design approach and have yielded a novel lentiviral vector with clinical promise for the treatment of X-CGD.

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein, we generated an expression plasmid encoding an optimized protein that produces S-pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein and 4 to 10-fold more specific than the widely used delta-19 S-pseudotyped lentiviral vectors. S-pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S-pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE2-expressing cells.

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