UCLA

We set out to improve upon current β-globin expressing lentiviral vectors (LV) designs by rationally reengineering human genomic sequences through the removal and/or addition of known elements. Although this approach generated improved LV designs, it is a low-throughput model for LV development, where large sections of DNA containing roughly-defined elements are simply inserted and/or removed from LVs and tested against a litany of criteria until a combination of well-performing elements are found (typically occurring over a span of years). To speed up LV development, we concurrently developed an advanced LV-engineering technology that can be broadly applied to the design of any gene therapy LV that requires high-level and lineage specific expression. Our Lentiviral Vector based, Massively Parallel Reporter Assay (LV-MPRA) allowed us to map the precise boundaries of lineage specific enhancers within large genomic regions (>16kb). The enhancer maps generated guided the assembly of novel enhancer combinations that when placed in a globin expression LV conferred robust transgene expression within a minimal DNA footprint. While our original “intelligent” design approach generated LV designs with improved performance (when compared to antecedent clinical LVs), vectors designed using the LV-MPRA based enhancer mapping approach possessed similar improvements in performance and were generated in only a fraction of the time (~3 months vs ~3 years). Moreover, LVs generated by either approach corrected the “Townes” mouse model of Sickle Cell Disease. The yields of this research offer improved “next generation” LVs for the gene therapy of hemoglobinopathies.