The Penn State Protein Ladder system for inexpensive protein molecular weight markers.

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10-50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.

Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.

Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous ßs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.

Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.