Background: Autologous hematopoietic stem cell transplantation combined with ex-vivo gene therapy is a promising approach in treating disorders of the hematopoietic system. Identifying combinations of strong lineage-specific control elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. Candidate enhancers or promoters must be tested against a litany of performance criteria until high-performing combinations can be found. A current limitation in designing enhancers of minimal-length is a lack of knowledge regarding the exact boundaries of “sequence intrinsic” enhancers (the actual DNA sequences that provide enhancer function) for a given cell type or state. Current technologies such as ChIP-Seq and its variants provide vague boundaries of enhancer location when specific combinations of DNA-protein binding can be observed within a region. Furthermore, technologies based on DNA-protein binding may fail to assist in identification of enhancer regions when proteins transiently bind to functional DNA sequences, perturb transcription by modifying local chromatin structure, and dissociate before they can be fixed in place by DNA-protein crosslinking.

Objective(s): We have developed a method termed LV-MPRA (Lentiviral Vector-based, Massively Parallel Reporter Assay), to generate targeted enhancer maps of the β-globin Locus Control Region (LCR) that provide boundaries of “sequence intrinsic” enhancers at near base-pair resolution. We use these maps to design lineage specific LVs and characterize their performance across multiple categories and then test the best LVs in a mouse model of Sickle Cell Disease.

Methods: Microarray generated DNAs spanning a targeted region (~16kb) are cloned into a lentiviral vector resulting in placement of, 1). A “Query” sequence upstream of a promoter and, 2). A barcode in both the 3’UTR of a reporter gene and upstream of a polyadenylation signal. The library is then transferred to a cell-line and the strength of a given “Query” sequence can be ascertained by quantifying the abundance of mRNA barcodes by next-generation sequencing (NGS). Those sequences identified to be within the highest percentiles of expression were concatenated and cloned into therapeutic expression vectors, which were tested in a mouse model of SCD for their ability to reverse disease phenotype.

Results: We employ LV-MPRA to elucidate the boundaries of the previously unknown “intrinsic enhancer” sequences of the β-globin Locus Control Region (LCR). We observe that enhancer activity peaks fall well within “classical” enhancer boundaries as defined by the literature. The LV-MPRA-guided constructs (termed 95 and 97.5) were then evaluated in the “Townes” mouse model of SCD to assess their ability to induce hematologic correction. At 16 weeks post bone marrow (BM) transplantation, average levels of %Hb βAS3 / total hemoglobin tetramers were found to be 0%, 31.8% and 39.6% in mice that received mock, 95, or 97.5 transduced BM, respectively. Moreover, statistically significant increases in total Hb levels and red blood cell counts were observed for 95 and 97.5 when compared to mice that received mock transduced BM.

Conclusion: We have harnessed the power of massively parallel automated DNA synthesis and NGS to simultaneously analyze thousands of synthetic DNA fragments in parallel to identify “sequence intrinsic” enhancers of the LCR at near base pair resolution.  These maps were used to generate novel LVs that ameliorated hematologic parameters defining the pathological phenotype of SCD in the mouse model of the disease. Case reports describing patients afflicted with both SCD and hereditary persistence of fetal hemoglobin often describe the clinical course as benign when HbF levels are 10% or higher (likely due to pancellular distribution of HBF). Thus, the in vivo percentages of βAS3-globin seen for 95 and 97.5 are at levels of expression expected to be therapeutic. These new LV designs should have advantages for clinical-scale production providing the highest level of gene transfer for the lowest amount of vector.