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SRX23858797: GSM8133059: R1-NHP-55-N-H3K4me3; Macaca mulatta; OTHER
1 ILLUMINA (Illumina HiSeq 4000) run: 4.5M spots, 678.2M bases, 276.2Mb downloads

External Id: GSM8133059_r1
Submitted by: CCSR 2110, Pediatrics & Genetics, Stanford School of Medicine
Study: Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human Factor IX variant in mice and rhesus macaques (CnT NHP)
show Abstracthide Abstract
We investigated long-term human coagulation Factor IX expression of a novel variant when delivered into mice and rhesus macaques and compare transduction efficiencies using two different AAV capsids. In hemophilic mice injected with KP1-packaged rAAV expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared to wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months post-administration, while one animal from each group lost huFIX mRNA and protein expression over time despite comparable vector copies. We investigated if epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques to those in mice injected with the same vector, the activating histone marks were starkly reduced in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques as well as the wide dose response variation observed in primates in both preclinical and human clinical trials. Overall design: Rhesus macaques were injected with rAAV packaged using different capsids, nuclei were isolated, and Cut&Tag analysis was performed
Sample: R1-NHP-55-N-H3K4me3
SAMN40287251 • SRS20678054 • All experiments • All runs
Organism: Macaca mulatta
Library:
Name: GSM8133059
Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Macaques were euthanized 3.5 months after rAAV injection and liver tissue from 4 lobes was flash-frozen. Frozen liver tissue was pulverized on dry ice and aliquoted. Equal amounts of pulverized liver tissue from each lobe were combined and ~100 mg of this pool were homogenized in RINO 1.5-ml Screw-Cap tubes filled with stainless steel beads and 1mL of NE buffer using a bead homogenizer (Next Advance Bullet Blender Storm - BBY24M) at speed 8 for 6 minutes. The homogenate was transferred into to a 50mL tube with 12mL of NE buffer and incubated on ice for 10 min. Nuclei were pelleted by a 5 min centrifugation at 600g, the supernatant was decanted, and the pellet was resuspended in 1mL of NE buffer. After passing nuclei through a 0.45um cell strainer quantitation was performed after Trypan Blue staining using an automated cell counter (Countess, Thermo Fisher) and diluted accordingly. 1e5 nuclei per target protein were used for Cut&Tag. Nuclei were mixed with activated Concanavalin A beads. After successive incubations with primary antibody (overnight) and secondary antibody (for 30 min) in antibody and Digitonin150 buffer respectively, the beads were washed with Digitonin150 buffer and resuspended in Digitonin300 buffer with 2.5uL of pA(G)-Tn5 for 1 hr and washed with the same buffer. Incubations were performed at room temperature in low-retention PCR strip tubes (Epicypher). Tagmentation was performed for 1 hr at 37C in Tagmentation buffer that provides MgCl2. Beads were washed with TAPS buffer and DNA material was released by adding SDS Release Buffer and incubating at 58C for 1hr. Quenching of SDS was performed by adding SDS Quench buffer and PCR was performed directly on this material. Universal P5 and indexed P7 primer solutions were used (see Supplemental Table 3 for sequences), and 17 cycles of PCR were performed. Clean-up was performed with AMPure beads, eluted in 15uL 0.1x TE buffer. Qubit and Bioanalyzer were used to verify library qualities before pooling samples for sequencing. The barcoded libraries were mixed to achieve equimolar representation aiming for a 10nM final concentration. Cut & Tag. Sequencing of the same library was performed twice (R1, R2), once with Illumina HiSeq 4000 and once with Illumina NovaSeq SP100, 150 cycles total per lane, 2x75 paired-end reads, depth of >15M reads per sample.
Runs: 1 run, 4.5M spots, 678.2M bases, 276.2Mb
Run# of Spots# of BasesSizePublished
SRR282481254,461,932678.2M276.2Mb2024-03-11

ID:
32162792

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