Dataset Information

Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

ABSTRACT: Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking.

SUBMITTER: Strobel B

PROVIDER: S-EPMC4554548 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

Strobel Benjamin B Miller Felix D FD Rist Wolfgang W Lamla Thorsten T

Human gene therapy methods 20150801 4

Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the ...[more]

PMID: 26222983

Similar Datasets

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.

Project description:Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

Dataset Information

Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

Publications

Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets