Project description:Adeno-associated viruses (AAVs) are non-enveloped ssDNA icosahedral T = 1 viruses used as vectors for clinical gene delivery. Currently, there are over 200 AAV-related clinical trials and six approved biologics on the market. As such new analytical methods are continually being developed to characterize and monitor the quality and purity of manufactured AAV vectors, these include ion-exchange chromatography and Direct Mass Technology. However, these methods require homogeneous analytical standards with a high molecular weight standard comparable to the mass of an AAV capsid. Described here is the design, production, purification, characterization, and the cryo-electron microscopy structure of an AAV1-VP3-only capsid that fulfills this need as a calibrant to determine capsid mass, charge, homogeneity, and transgene packaging characteristics.
Project description:With more than 130 clinical trials and eight approved gene therapy products, AAVs stand as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell‑derived RNAs, such as micro‑RNAs, has not been previously investigated. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co‑purified with the rAAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non encapsidated into AAV particles. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.
Project description:Adeno-associated virus (AAV) vectors generated with five different capsids were intravenously injected into human livers undergoing normothermic machine perfusion (NMP). To assess the tropism of the five AAV vectors in the human livers, single-cell suspensions of hepatocytes and non-parenchymal cells (NPCs) were analyzed by single-cell RNA sequencing (scRNAseq). Differentially expressed genes were identified in AAV vector-transduced hepatocytes and unique cell populations.
Project description:BackgroundClinical use of genotype data requires high positive predictive value (PPV) and thorough understanding of the genotyping platform characteristics. BeadChip arrays, such as the Global Screening Array (GSA), potentially offer a high-throughput, low-cost clinical screen for known variants. We hypothesize that quality assessment and comparison to whole-genome sequence and benchmark data establish the analytical validity of GSA genotyping.MethodsTo test this hypothesis, we selected 263 samples from Coriell, generated GSA genotypes in triplicate, generated whole genome sequence (rWGS) genotypes, assessed the quality of each set of genotypes, and compared each set of genotypes to each other and to the 1000 Genomes Phase 3 (1KG) genotypes, a performance benchmark. For 59 genes (MAP59), we also performed theoretical and empirical evaluation of variants deemed medically actionable predispositions.ResultsQuality analyses detected sample contamination and increased assay failure along the chip margins. Comparison to benchmark data demonstrated that > 82% of the GSA assays had a PPV of 1. GSA assays targeting transitions, genomic regions of high complexity, and common variants performed better than those targeting transversions, regions of low complexity, and rare variants. Comparison of GSA data to rWGS and 1KG data showed > 99% performance across all measured parameters. Consistent with predictions from prior studies, the GSA detection of variation within the MAP59 genes was 3/261.ConclusionWe establish the analytical validity of GSA assays using quality analytics and comparison to benchmark and rWGS data. GSA assays meet the standards of a clinical screen although assays interrogating rare variants, transversions, and variants within low-complexity regions require careful evaluation.
Project description:Cell-mediated immune (CMI) responses to adeno-associated virus (AAV) can lead to both tissue damage and loss of therapeutic transgene expression. Identifying robust biomarkers of CMI and understanding CMI mechanisms can be help to helpful in clinical practice and in advancement of AAV gene therapies. The present work evaluated PBMCs from non-human primates (NHP) before and 14 days following immunization with AAV9 capsid. PBMCs were stimulated in vitro with AA9 capsid peptides to evaluate CMI responses by IFN- ELISPOT, intracellular cytokines and activation markers by flow cytometry, secreted cytokines by ELISAMSD, and transcriptional responses by RNAseq. AAV peptide stimulation produced a robust IFN- ELISPOT response at 14 days. Flow cytometry revealed an increase in IFN- positive CD4 and CD8 cells, IL2 positive CD4 cells, and TNF positive CD4 cells. An increase in the CXCR3 ligands IP-10 and I-TAC were detected both inas secreted proteins. The most robust changes in response to AAV stimulation and strongest correlations to ELISPOT response were revealed by RNAseq, including transcripts encoding IFN- and transcripts encoding , IP-10 and I-TAC , and many downstream transcripts and several IFN-independent pathways. These data provide evidence that a gene signature, or CXCR3 ligand transcripts or protein could serve as robust and sensitive alternatives to ELISPOT for detection of CMI, andCMI and warrant further benchmarking in human samples.
Project description:We used AAV as a vector to deliver hGRβ to C57BL/6 mouse liver. We collected liver samples for microarray analysis to investigate any phenotype as well as the underlying specific signaling pathway. In particular, we would like to determine if and how hGRβ overexpression in liver affects mGRαâs gene transcription profile in C57BL/6 mice. Three replicates for each group: untreated WT liver, AAV-GFP treated liver, and AAV-hGRB treated liver.
Project description:HBV-transgenic mice were treated i.v. with 1x10e11 particles AAV serotype 8 vectors expressing different RNAi-triggers targeting the HBV transcripts. To determine possible toxicities caused by the different vectors (including off-target activity against endogenous genes) we performed a whole transcriptome analysis of RNA of livers harvested 15 days after injection. 4 replicates per treatment group were analyzed. As controls, one group of mice was treated with 0.9% saline (mock) and another group of animals was treated with an empty AAV8 vector (1x10e11 particles i.v.).
Project description:Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. We used a primate specific capsid, AAV-LK03, and demonstrated that the limitation of this capsid towards transduction of mouse cells was unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enabled efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately led to rationale selection of AAV capsids for use in humans.
Project description:Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. We used a primate specific capsid, AAV-LK03, and demonstrated that the limitation of this capsid towards transduction of mouse cells was unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enabled efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately led to rationale selection of AAV capsids for use in humans.