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.

This work demonstrates the possibility of producing purified high-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors.

eThe clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a speciesspecific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype-and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.A deno-associated virus (AAV) vectors attract great attention as a promising tool for a wide range of applications in gene therapy. The process of cell transduction by recombinant AAVs (rAAVs) has been studied in detail, and cellular receptors responsible for the virus entry have been identified. Most of these studies were accomplished in cell culture (1-3) without taking into account the exposure of rAAVs to components of body fluids in the in vivo situation. Interestingly, in many cases, protein classes having specific posttranslational modifications, such as ␣-2,3 and ␣-2,6 sialic acids, N-linked glycoproteins, or heparan sulfate proteoglycan, were identified as primary cell receptors for efficient rAAV transduction (4-6). These posttranslational modifications are common between mammalian species, giving hope to the possibility that rAAV efficiency could be similar across species and that animal data are predictive of the human situation.Nevertheless, some recent data indicate that interactions of cellular receptors or blood proteins with rAAVs can be species specific. Thus, adeno-associated virus vector 3 (rAAV-3), which efficiently transduces human hepatocytes through the hepatocyte growth factor receptor (HGFR), failed to transduce murine hepatocytes, suggesting that AAV-3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction (7-9). In human and dog blood, but neither mouse nor monkey blood, galectin 3 binding protein (G3BP) in...

Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an in vivo role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials.