Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography

Scherr, Michaela; Battmer, Karin; Eder, Matthias; Schüle, Silke; Hohenberg, Heinz; Ganser, Arnold; Grez, Manuel; Blömer, Ulrike

doi:10.1038/sj.gt.3301848

Cited by 72 publications

(48 citation statements)

References 28 publications

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“…Moreover, omitting the use of serum in the culture media eliminates the risk of contamination with adventitious agents and minimizes regulatory concerns. The recovery of infective viral particles (53%) by heparin affinity chromatography was comparable to those reported for lentiviral vectors using anion exchange chromatography (Scherr et al, 2002;Slepushkin et al, 2003;Yamada et al, 2003) and to those obtained with oncoretroviral vectors produced in the presence of 10% FBS (Segura et al, 2005(Segura et al, , 2006b. As with size exclusion chromatography and anion exchange chromatography, treatment of lentiviral vector stocks with DNAse will be required to remove residual contaminating nucleic acids .…”

Section: Discussionsupporting

confidence: 73%

“…Moreover, elimination of serum proteins and producer cellderived contaminants from vector supernatants has shown to prevent local inflammatory and immune responses in vivo (Baekelandt et al, 2003;Scherr et al, 2002) and to increase transduction efficiencies ex vivo (Yamada et al, 2003). For this reason, harvested vector supernatants must undergo a series of processing steps aimed at improving the potency and purity of the vector preparation (for review see Segura et al (2006a)).…”

Section: Introductionmentioning

confidence: 99%

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Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

Segura

Garnier

Durocher

et al. 2007

Biotech & Bioengineering

119

107

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The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaledup from shake flaks to a 3-L bioreactor from which 10 10 IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant. Biotechnol. Bioeng. 2007;98: 789-799.

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Section: Discussionsupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

Segura

Garnier

Durocher

et al. 2007

Biotech & Bioengineering

119

107

View full text Add to dashboard Cite

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“…Recently, methods based on anion exchange chromatography of HIV-1 vectors pseudotyped with VSV.G and baculovirus glycoprotein 64 have been successfully established. [81][82][83] Titer determination. To standardize the number of viral particles administered to patients, it is crucial to accurately titrate the virus for each individual vector production.…”

Section: Downstream Processing Of Lentiviral Vectorsmentioning

confidence: 99%

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics

2007

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Lentiviral vectors have emerged as promising tools for both gene therapy and immunotherapy purposes. They exhibit several advantages over other viral systems in that they are less immunogenic and are capable of transducing a wide range of different cell types, including dendritic cells (DC). DC transduced ex vivo with a whole range of different (tumor) antigens were capable of inducing strong antigen-specific T-cell responses, both in vitro and in vivo. Recently, the administration of lentiviral vectors in vivo has gained substantial interest as an alternative method for antigenspecific immunization. This method offers a number of advantages over DC vaccines as the same lentivirus can in principle be used for all patients resulting in a significantly reduced cost and requirement for considerably less expertise for the generation and administration of lentiviral vaccines. By selectively targeting lentiviral vectors to, or restricting transgene expression in certain cell types, selectivity, safety and efficacy can be further improved. This review will focus on the use of direct administration of lentiviral vectors encoding tumor-associated antigens (TAA) for the induction of tumor-specific immune responses in vivo, with a special focus on problems related to the generation of large amounts of highly purified virus and specific targeting of antigenpresenting cells (APC).

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“…Most of these immune responses could be blunted by the use of modified vector backbones (eg by using 'gutted' vectors), muscle-specific promoters (in either Ad or rAAV vectors), or by using ultrapurified vector preparations (eg HPLC purification of lentiviral vectors). 5,6,23,24,43 However, we observed that muscles transduced with the L-CK6-ntsLacZ still showed decreased b-galactosidase activity with increasing time (data not shown), while dystrophin expression from the L-CMV-DH2-R19 vector appeared stable (Figure 2b).…”

Section: Discussionmentioning

confidence: 91%

“…Vector-mediated immune responses have previously been observed in a variety of tissues, including muscle, following injection with either Ad, rAAV, or lentiviral vectors. 23,24,43 These responses can be directed against vector proteins, foreign transgene expression, or impurities in the viral preparation. Most of these immune responses could be blunted by the use of modified vector backbones (eg by using 'gutted' vectors), muscle-specific promoters (in either Ad or rAAV vectors), or by using ultrapurified vector preparations (eg HPLC purification of lentiviral vectors).…”

Section: Discussionmentioning

confidence: 99%

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

Kimura

Fall

et al. 2005

Gene Ther

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Gene therapy for Duchenne muscular dystrophy (DMD) will require sustained expression of therapeutic dystrophins in striated muscles. Lentiviral vectors have a relatively large transgene carrying capacity and can integrate into nondividing cells. We therefore explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells. These vectors successfully transferred a minidystrophin expression cassette into mdx muscles, and minidystrophin expression persisted and prevented subsequent muscle fiber degeneration for at least 6 months. However, only low to moderate levels of skeletal muscle transduction could be obtained by intramuscular injection of the highest currently available lentiviral doses. Using cultured cells, the lentiviral vectors effectively transduced proliferating and terminally differentiated muscle cells, indicating that cell cycling is not essential for transduction of myogenic cells. We further showed that lentiviral vectors efficiently transduced both primary myoblasts and multipotent adult progenitor cells (MAPCs) in vitro, and the cells persistently expressed transgenes without any obvious toxicity. When mdx primary myoblasts were genetically modified with minidystrophin vectors and transplanted into mdx skeletal muscles, significant numbers of dystrophin-expressing myofibers formed. Finally, we showed that a short, highly active CK6 regulatory cassette directed muscle-specific activity in the context of the lentiviral vectors. The ability of lentiviral vectors to transduce myogenic progenitors using a minidystrophin cassette regulated by a muscle-specific promoter suggests that this system could be useful for ex vivo gene therapy of muscular dystrophy.

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Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography

Cited by 72 publications

References 28 publications

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

Lentiviral vectors for cancer immunotherapy: transforming infectious particles into therapeutics

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

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