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

Abstract

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 CD34+ cells 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 × 104 to 1.0 × 106 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, 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 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

Delivery of large transgene cassettes by foamy virus vector

“…At that size, FVVs have only 10-fold reduced titre. Since lentivirus and FVVs with small inserts can be produced to similar titres 31 , 32 , it appears that FVVs have an advantage over LVVs of being able to deliver approximately twice as much cargo as LVVs at useful titres. This information will be valuable for approaches where the stable delivery of multiple and/or large transgene cassettes is required.…”

FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs

“…These modifications rendered FV vectors as efficient as lentiviral vectors producing 10 × 10 7 TU/mL of crude supernatant [44]. Finally, FV supernatants can be concentrated and purified in closed systems using affinity chromatography with POROS-Heparin columns, followed by Tangential Flow Filtration and ultracentrifugation [50] or size exclusion chromatography using CaptoCore columns followed by sepharose-heparin affinity chromatography and ultracentrifugation [51]. Both purification approaches are performed in closed systems compliant with cGMP and yield significantly pure preparations.…”

Gene therapy approaches to functional cure and protection of hematopoietic potential in HIV infection

“…Induced pluripotent stem cells (iPSCs) may also be candidates for anti-HIV gene transfer. iPSCs can be generated from the patients' somatic cells, differentiate to any cells in vitro, and are expected to be utilized for the treatment of a broad range of genetic diseases [119][120][121][122]. While CD34 + cells engraft in the bone marrow following transplantation and differentiate to hematopoietic cells in vivo, iPSCs may be more convenient for in vitro hematopoiesis than CD34 + cells because of their ease of culture [123].…”