Dataset Information

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

ABSTRACT: We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m².

SUBMITTER: Marichal-Gallardo P

PROVIDER: S-EPMC10116406 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Publications

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

Marichal-Gallardo Pavel P Börner Kathleen K Pieler Michael M MM Sonntag-Buck Vera V Obr Martin M Bejarano David D Wolff Michael W MW Kräusslich Hans-Georg HG Reichl Udo U Grimm Dirk D

Human gene therapy 20210330 17-18

We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is ...[more]

PMID: 33554722

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Project description:Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

Dataset Information

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

Publications

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

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