Journal: iScience

Article Title: Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality.

doi: 10.1016/j.isci.2023.106487

Figure Lengend Snippet: Figure 4. Novel AAV vector production system is applicable for other serotypes (A) Western blot of replicase (Rep) and capsid (Cap) proteins during adeno-associated virus (AAV) vector production for various serotypes in HEK293 cells. Rep, Cap, and b-actin proteins were detected using anti-Rep, anti-Cap, and anti- b-actin antibodies, respectively. (B–E) AAV vector yield in various serotypes for the Tet-Cap system. Fold difference of AAV vector yield in AAV7 (B), AAV8 (C), AAV9 (D), and AAVrh10 (E) after a change in medium and doxycycline stimulation at 12 h after transfection. Data were normalized with the value of the normal control (RC) samples. (F–I) Western blot of Cap proteins after immunoprecipitation for various serotypes. The same titer of AAV vector (1 3 109– 4 3 109 vg/sample) calculated with qPCR was subjected to immunoprecipitation using ADK8 (AAV7, AAV8, and rh10) and ADK8/9 (AAV9) antibodies, as well as protein A/G magnetic beads, before western blotting. The left-hand figures show representative western blotting images, and the right-hand figures the fold difference of band intensities for VP1, VP2, and VP3 proteins between RC and Tet-Cap samples in each panel. These panels indicate the data for AAV7 (F), AAV8 (G), AAV9 (H), and AAVrh10 (I), respectively. Experiments were independently performed at least three times for statistical analysis. Asterisks in each panel indicate the following: * = p< 0.05, ** = p< 0.01. Error bars indicate the standard error of the mean.

Article Snippet: AAV serotypes 1, 7, 8, 9, and rh10 capsid (Cap) plasmids (pAAV2/1, pAAV2/7, pAAV2/8, pAAV2/9n, and pAAV2/rh10) were gifted by James M. Wilson (Addgene plasmids: 112862, 112863, 112864, 112865, and 112866).

Techniques: Plasmid Preparation, Western Blot, Virus, Transfection, Control, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Localized in vivo gene editing of murine cancer-associated fibroblasts

doi: 10.1101/2024.07.11.603114

Figure Lengend Snippet: (A) NIH/3T3.Cas9 cells were transduced with three different AAVs as outlined in all carrying the same hU6-gThy1-mCherry cassette but generated with three different capsids (AAV1, 7m8, DJ). Left, flow cytometry plots depicting mCherry expression in NIH/3T3.Cas9 cells as a readout for transduction efficiency of AAVs after 96 h at an MOI of 1e5. Right, flow cytometry overlay plots of those NIH/3T3.Cas9 cells showing KO efficiency of the AAVs with different capsids at day 6 post AAV transduction. One of two representative experiments is shown. (B) In vivo gating scheme to identify tumor fibroblasts: DAPI - CD31 - CD45 - E-Cadherin - MCAM - CD90.2 + PDPN + EGFP + in Pdgfra -Cas9-EGFP mice. For Col1a2 CreERT2/+ ; Tgfbr2 fl/fl mice in and , tumor fibroblasts were gated as DAPI - CD31 - CD45 - E-Cadherin - MCAM - CD90.2 + PDPN + . (C) (Top) Flow cytometry contour plots of EGFP expression in tumor fibroblasts as gated in (B) on day 14 after 2.5e5 HY19636 pancreatic tumor cell s.c. injection in Pdgfra -Cre; R26 -Cas9-EGFP +/+ or Pdgfra -CreERT2 +/wt ; R26 -Cas9-EGFP +/+ mice (both referred to as Pdgfra -Cas9-EGFP mice). Pdgfra -CreERT2 +/wt ; R26 -Cas9-EGFP +/+ mice were injected with 2 mg of tamoxifen (TAM) on 5 consecutive days one week prior to tumor engraftment to induce Cas9-EGFP expression. (Bottom) Contour plots of fibroblast markers CD90.2 and PDPN expression in DAPI - EGFP + cells. (D) Pictures of subsequent injections of AAV and tumor cells on consecutive days to generate local gene KO in tumor fibroblasts. Local AAV injection is marked by tattoo paste using a 31 gauge needle on day -1 around wheal from AAV injection. Tattoo pricks are visible on day 0 and used as a guide for tumor cell injection. (E) Flow cytometry contour plots of EGFP+ tumor fibroblasts from Pdgfra- Cas9-EGFP mice injected s.c. with 1e10 vg AAV-gThy1 on day -1 and 5e5 YUMM5.2 tumor cells on day 0. Tumors were excised on day 7 and (left) mCherry expression in tumor fibroblasts (gated as outlined in (B)) was measured as a readout for transduction efficiency and (right) surface level of CD90.2 was measured as a readout for knockout efficiency by the different AAV capsids. (F) Test of different scAAV1 doses for transduction and KO in tumor fibroblasts. Pdgfra -Cas9-EGFP mice were injected s.c. on day -1 with 1e10 or 3e10 vg of scAAV1-gThy1-mCherry (self-complementary AAV carrying gRNA targeting Thy1 and an mCherry ORF). On day 0, 5e5 YUMM5.2 cells were injected s.c. at site of prior AAV injection. Tumors were harvested on day 7 and transduction and Thy1 knockout efficiency was assessed. Top, flow cytometry plots of all tumor fibroblasts (gated as outlined in (B)) showing mCherry expression. Bottom, flow cytometry plots of all tumor fibroblasts for CD90.2 surface expression. Right, quantification of transduction and KO efficiency at two different doses of scAAV1-gThy1. Data are mean ± s.d. and significance was tested using one-way ANOVA with Tukey’s multiple comparison test. ns, non-significant. ****p<0.0001. (G) Flow cytometry overlay plot of surface TGFBR2 expression on NIH/3T3.Cas9 cells transduced with or without scAAV1-gTgfbr2 at an MOI of 1e6 and analyzed 72h later. MOI, multiplicity of infection. scAAV, self-complementary adeno-associated virus. s.c., subcutaneously. vg, viral genome.

Article Snippet: The helper plasmid (pHelper, Takara, #6234) was used in conjunction with one of three different RepCap plasmids (7m8, Addgene, #64839; pAAV-DJ, Cell Biolabs, VPK-420-DJ; pAAV2/1, Addgene, #112862), and a self-complementary AAV cargo vector (see design below) to package vector genomes into double-stranded AAV particles.

Techniques: Transduction, Generated, Flow Cytometry, Expressing, In Vivo, Injection, Knock-Out, Comparison, Infection, Virus