production-purification-baculovirus-for-gene-therapy

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

purification-of-platelets-from-mouse-blood

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

process-development-for-production-purification-adeno-associated

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

Cincinnati Children's Hospital Medical Center

University of Cincinnati College of Medicine

Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 10to 1.0 × 10infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1-2 × 10IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency.

Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26-29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications.

Purification of baculovirus vectors using heparin affinity chromatography.

Sickle cell anemia (SCA) is caused by a point mutation in the β-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αβ integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αβ Fibγ mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.

Elimination of the fibrinogen integrin αβ-binding motif improves renal pathology in mice with sickle cell anemia.

Sickle cell disease (SCD) is an inherited monogenic red blood cell disorder affecting millions worldwide. SCD causes vascular occlusions, chronic hemolytic anemia, and cumulative organ damage such as nephropathy, pulmonary hypertension, pathologic heart remodeling, and liver necrosis. Coagulation system activation, a conspicuous feature of SCD that causes chronic inflammation, is an important component of SCD pathophysiology. The key coagulation factor, thrombin (factor IIa [FIIa]), is both a central protease in hemostasis and thrombosis and a key modifier of inflammation. Pharmacologic or genetic reduction of circulating prothrombin in Berkeley sickle mice significantly improves survival, ameliorates vascular inflammation, and results in markedly reduced end-organ damage. Accordingly, factors both upstream and downstream of thrombin, such as the tissue factor-FX complex, fibrinogen, platelets, von Willebrand factor, FXII, high-molecular-weight kininogen, etc, also play important roles in SCD pathogenesis. In this review, we discuss the various aspects of coagulation system activation and their roles in the pathophysiology of SCD.

Md Nasimuzzaman

Division of Experimental Hematology and Cancer Biology

Cincinnati Children's Hospital Medical Center

Production and Purification of Baculovirus for Gene Therapy Application

Purification of Platelets from Mouse Blood

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Md Nasimuzzaman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Experimental Hematology and Cancer Biology

Cincinnati Children's Hospital Medical Center

Production and Purification of Baculovirus for Gene Therapy Application

Purification of Platelets from Mouse Blood

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Division of Experimental Hematology and Cancer Biology