From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

The importance of mesenchymal-epithelial interactions for normal development of the pancreas was recognized in the early 1960s, and mesenchymal signals have been shown to control the proliferation of early pancreatic progenitor cells. The mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce the normal number of differentiated pancreatic cells are not fully understood. Here, we demonstrate that the mesenchyme positively controls the final number of ␤-cells that develop from early pancreatic progenitor cells. In vitro, the number of ␤-cells that developed from rat embryonic pancreatic epithelia was larger in cultures with mesenchyme than without mesenchyme. The effect of mesenchyme was not due to an increase in ␤-cell proliferation but was due to increased proliferation of early pancreatic duodenal homeobox-1 (PDX1)-positive progenitor cells, as confirmed by bromodeoxyuridine incorporation. Consequently, the window during which early PDX1؉ pancreatic progenitor cells differentiated into endocrine progenitor cells expressing Ngn3 was extended. Fibroblast growth factor 10 mimicked mesenchyme effects on proliferation of early PDX1؉ progenitor cells and induction of Ngn3 expression. Taken together, our results indicate that expansion of early PDX1؉ pancreatic progenitor cells represents a way to increase the final number of ␤-cells developing from early embryonic pancreas. Diabetes 56:1248-1258, 2007 E pithelium-mesenchyme interactions play a crucial role during organogenesis. They are mediated at least in part by soluble factors produced by the mesenchyme and acting on the epithelium (1). Evidence points to a crucial role for epithelialmesenchymal interactions in cell proliferation and differentiation during pancreatic development (2). However, the mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce a normal number of differentiated pancreatic cells are not fully understood.The mature pancreas contains endocrine islets composed of cells producing hormones, such as insulin (␤-cells) and glucagon (␣-cells), and exocrine tissue composed of acinar cells producing enzymes (e.g., carboxypeptidase-A) secreted into the intestine. The pancreas originates from the dorsal and ventral regions of the foregut endoderm. Recently, important findings have shed light on the processes controlling pancreatic endocrine cell development. Studies of genetically engineered mice identified a hierarchy of transcription factors regulating pancreas organogenesis and islet-cell differentiation (3-5). The endodermal region committed to a pancreatic fate first expresses transcription factor pancreatic duodenal homeobox-1 (Pdx1). Pdx1 is detected in mouse embryos on embryonic day 8.5 (E8.5) (E9 in rats) in early pancreatic progenitors. During adulthood, Pdx1 expression becomes largely confined to ␤-cells, where it activates insulin gene transcription (6). Disruption of the Pdx1 gene in mice or human leads to pancreatic agenesis (7,8). These data indicate that P...

No abstract

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.