Dataset Information

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors for In Vivo Transduction.

ABSTRACT: Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed, heavier particles found in rAAV preparations have traditionally been ignored due to their reported low in vitro transduction efficiency. In this study, the biological properties of regular and high-density rAAV serotype 8 vectors, rAAVRD and rAAVHD, were systemically compared. Results demonstrated that both rAAVRD and rAAVHD exhibited similar DNA packaging profiles, while rAAVHD capsids contained fewer VP1 and VP2 proteins, indicating that the rAAVHD particles contained a higher DNA/protein ratio than that of rAAVRD particles. Dynamic light scattering and transmission electron microscopy data revealed that the diameter of rAAVHD was smaller than that of rAAVRD. In vitro, rAAVHD was two- to fourfold less efficient in transduction compared with rAAVRD. However, the transduction performance of rAAVHD and rAAVRD was similar in vivo. No significant difference in neutralizing antibody formation against rAAVRD and rAAVHD was observed, suggesting that the surface epitopes of rAAVRD and rAAVHD are congruent. In summary, the results of this study demonstrate that rAAVRD and rAAVHD are equally competent for in vivo transduction, despite their difference in vitro. Therefore, the use of rAAVHD vectors in human gene therapy should be further evaluated.

SUBMITTER: Wang Q

PROVIDER: S-EPMC5178025 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors for In Vivo Transduction.

Wang Qizhao Q Firrman Jenni J Wu Zhongren Z Pokiniewski Katie A KA Valencia C Alexander CA Wang Hairong H Wei Hongying H Zhuang Zhenjing Z Liu LinShu L Wunder Stephanie L SL Chin Mario P S MP Xu Ruian R Diao Yong Y Dong Biao B Xiao Weidong W

Human gene therapy 20160822 12

Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed, heavier particles found in rAAV preparations have traditionally been ignored due to their reported low in vitro transduction efficiency. In this study, the biological properties of regular and high-density rAAV serotype 8 vectors, rAAVRD and rAAVHD, were systemically compared. Results demonstrated that both rAAVRD and ...[more]

PMID: 27550145

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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.

Project description:Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

Dataset Information

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors for In Vivo Transduction.

Publications

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors for In Vivo Transduction.

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