Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Strategy for therapeutic targeting of the Apoa1 locus for liver gene therapy AAV-CRISPR encodes a gRNA for targeting the Apoa1 3′ UTR, and SaCas9 driven by a liver-specific promoter (HLP). The gRNA target site (red) and protospacer adjacent motif (PAM) sequence (blue) in the Apoa1 3′ UTR are shown. AAV-Donor contains the final coding exon of Apoa1 (exon 4) fused to a 2A ribosomal skipping sequence upstream of a therapeutic gene and synthetic polyadenylation signal (pA), flanked by homology arms (HR) to the Apoa1 locus. Following correct integration by HDR, the Apoa1 promoter drives the expression of a bicistronic mRNA consisting of Apoa1 and therapeutic transgene. Translation results in expression of apoA1 with a C-terminal 2A epitope tag, as well as the therapeutic protein including an N-terminal proline. Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: CRISPR, Sequencing, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Targeted integration and expression from the Apoa1 locus in vivo (A) Adult C57BL/6J mice were intraperitoneally injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor template (5 × 10 11 GC) encoding a far-red fluorescent protein (mKate2). Control mice were injected with AAV-GFP (5 × 10 11 GC). Livers and plasma were harvested for analysis at 12 weeks post-injection. (B) PCR from liver showing integration of AAV-Donor into the Apoa1 locus. Two main products were observed: correct HDR (1,139 bp) and NHEJ insertion (2,031 bp). The forward primer binds to the Apoa1 locus upstream of the 5′ HR and reverse primer binds to the mKate2 coding sequence. Minus (–) indicates water-only control. (C) Frequency of indel formation in the Apoa1 3′ UTR by deep sequencing. (D) Frequency of Apoa1 alleles with NHEJ insertions of AAV genomes by ddPCR. (E) Frequency of correct HDR targeting of AAV-Donor by ddPCR. (F) Representative immunohistochemistry for mKate2-FLAG (brown cells) in Apoa1 -targeted mice. Scale bar is 100 μm. (G) Quantification of FLAG positive hepatocytes relative to total nuclei per field. (H) Western blot of mKate2-FLAG in liver lysates with β-tubulin (β-tub) as a loading control. (I and J) Western blot analysis of 2A-tagged (I) and total apoA1 (J) in plasma with alpha-1 antitrypsin (aat) as loading control. Four representative mice per group are shown in western blots. (K) Densitometry analysis of mKate2-FLAG in liver lysates relative to β-tub loading control. Densitometry analysis of apoA1-2A (L) and apoA1 (M) in plasma relative to aat loading control. Data are shown as mean ± standard deviation (n = 5; n = 4 in densitometry analyses), with significance determined by one-way ANOVA followed by Tukey test. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Expressing, In Vivo, Injection, CRISPR, Sequencing, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Improved targeting of the Apoa1 locus in neonatal mice injected at P4 (A) Experimental design and AAV-Donor scheme. C57BL/6J mice were subcutaneously injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor template (5 × 10 11 GC) encoding a far-red fluorescent protein (mKate2) at P4 and liver and plasma were evaluated for the expression of the mKate2 protein at 20 weeks post-injection. Control mice were injected with AAV-GFP (5 × 10 11 GC). (B) Integration PCR on liver showed two main products corresponding to correct HDR (1,139 bp) and NHEJ insertion (2,031 bp) of AAV-Donor at the Apoa1 locus. Minus (–) indicates water-only control. (C) Frequency of Apoa1 alleles with NHEJ insertions of AAV genomes by ddPCR. (D) Frequency of correct HDR targeting of AAV-Donor by ddPCR. (E) Indel formation in the 3′ UTR of Apoa1 by ICE analysis. (F) Representative immunohistochemistry for mKate2-FLAG (brown cells) in Apoa1 -targeted mice. Scale bar is 100 μm. (G) Quantification of FLAG-positive hepatocytes relative to total nuclei per field. (H) Western blot of mKate2-FLAG in liver lysates with β-tub as a loading control. (I and J) Western blot analysis of 2A-tagged (I) and total apoA1 (J) in plasma with aat as loading control. Aat shows a slight difference in protein expression based on sex: lanes 1, 4, 7, 8, 9, 10, 13, 14, and 15 are male mice; lanes 2, 3, 5, 6, 11, 12, and 16 are female mice. Four representative mice per group are shown in western blots. (K) Densitometry analysis of mKate2-FLAG in liver lysates relative to β-tub loading control. Densitometry analysis of apoA1-2A (L) and apoA1 (M) in plasma relative to aat loading control. Data are shown as mean ± standard deviation (n = 5; n = 4 in densitometry analyses), with significance determined by one-way ANOVA followed by Tukey test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Injection, CRISPR, Expressing, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Sustained expression of factor IX over time in Apoa1 -targeted mice (A) AAV-Donor scheme. (B) Adult C57BL/6J mice were intraperitoneally injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor (5 × 10 11 GC) encoding human FIX . Control mice were injected with AAV-GFP (5 × 10 11 GC). Plasma was collected every 2 to 4 weeks up to 24 weeks post-injection for analysis of human FIX protein levels. (C and D) Western blot analysis of 2A-tagged (C) and total apoA1 (D) in plasma isolated at 24 weeks post-injection, with aat as loading control. Five representative mice per group are shown in western blots. (E and F) Densitometry analysis of apoA1-2A (E) and apoA1 (F) in plasma relative to aat loading control. (G) Quantitative measurement of plasma FIX over time by ELISA (green line, control; blue line, Donor; red line, CRISPR + Donor mice). Data are shown as mean ± standard deviation (n = 5 in densitometry analyses; n = 6 in FIX ELISA). A one-way and two-way ANOVA followed by Tukey test were used respectively for densitometry analyses (E and F) and FIX ELISA (G). ∗p < 0.05 CRISPR + Donor versus Donor; ∗∗p < 0.01 CRISPR + Donor versus Donor; ∗∗∗p < 0.001 CRISPR + Donor versus Donor, # p < 0.0001 Control versus Donor and CRISPR + Donor, ∗∗∗∗p < 0.0001. (A and B) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Expressing, Injection, CRISPR, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Reduction of plasma lipids in a mouse model of hypercholesterolemia through Apoa1 -targeting (A) AAV-Donor scheme. (B) P4 Apoe −/− male pups were subcutaneously injected with AAV-CRISPR (5 × 10 11 GC) and an AAV-Donor (5 × 10 11 GC) encoding human APOE or saline (control). Mice were fed a western diet starting at weaning for 20 weeks. Plasma was collected every 2 to 4 weeks up to 23 weeks of age. (C) Integration PCR on liver DNA showed two main products corresponding to HDR (1,289 bp) and NHEJ (2,213 bp) insertion of AAV-Donor in the Apoa1 cut site. Minus (–) indicates a water-only PCR control. (D–G) Western blot analysis of 2A-tagged apoA1 (D), APOE (E), total apoA1 (F), and apoB-48 and apoB-100 (G) in plasma isolated at endpoint, with aat as loading control. Eight representative mice per group are shown in western blots. (H–K) Densitometry analysis of apoA1-2A (H), APOE (I), apoA1 (J), and apoB-48 (K) in plasma relative to aat loading control. (L and M) Plasma total cholesterol (L) and triglycerides (M) measurement over time (green line, control; red line, CRISPR + Donor mice). Data are shown as mean ± standard deviation (n = 8 for densitometry analyses; n = 11 control and 9 CRISPR + Donor mice for lipid analyses). Significance was determined by a two-tailed Student’s t test in densitometry analyses (H–K). A two-way ANOVA followed by Bonferroni test was used for plasma lipid analyses in (L) and (M). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (A and B) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Injection, CRISPR, Western Blot, Isolation, Standard Deviation, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Correction of HT-I through targeted integration at the Apoa1 locus (A) Adult Fah −/− mice were intraperitoneally injected with either saline (control) or AAV-CRISPR plus an AAV-Donor encoding a human FAH transgene (5 × 10 11 GC each; CRISPR + Donor). NTBC was withdrawn at 7 days post-injection (time 0). Body weight was monitored over time up to 40 days and liver and plasma samples were collected for analyses. (B) Body weight ratios normalized to time zero (red line, CRISPR + Donor; green line, control mice). (C) Kaplan-Meier curve showing disease-free survival. Mice were euthanized upon loss of >20% body weight (red line, CRISPR + Donor; green line, control mice). One control mouse was found dead at day 30. (D) Western blot analysis of FAH in liver lysates with β-tub as a loading control. Plus (+) indicates a wild-type mouse liver as a positive control for endogenous Fah levels. (E) Representative FAH and hematoxylin and eosin staining in liver from control mice. (F) Representative FAH and hematoxylin and eosin staining in liver from CRISPR + Donor mice. Red squares are amplified in the images on the right. Scale bar is 100 μm. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Injection, CRISPR, Western Blot, Positive Control, Staining, Amplification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice

doi: 10.1074/mcp.M116.065474

Figure Lengend Snippet: Validation of the differentially expressed proteins that were identified by MALDI-TOF/TOF-MS. Seven proteins (HSP90B1, beta-actin, albumin, APOA1, CALR, FABP5, and PRDX6) were randomly selected from the differently expressed proteins that were identified by MALDI-TOF/TOF-MS and were validated in transgenic and nontransgenic males (A) and females (B) by Western blot assay. Wt: the liver tissue of wild-type mice; P: the peritumor tissue of transgenic mice; T: the tumor tissue of transgenic mice; Non-Tg, C57BL/6J wild-type nontransgenic mice; Tg: transgenic mice. The numbers indicate different individuals. Bradford reagent was used as the loading control.

Article Snippet: Primary and secondary antibodies: anti-phoshpo-MEK-1/2 polyclonal antibody (diluted 1:1000, Elabscience, Wuhan, China), anti-phoshpo-ERK1/2 monoclonal antibody (diluted 1:2000, Cell Signaling Technology, MA), anti-phoshpo-PI3 Kinase polyclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-phoshpo-Akt monoclonal antibody (diluted 1:2000, Cell Signaling Technology), anti-phoshpo-mTOR monoclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-APOA1 monoclonal antibody (diluted 1:1000, Proteintech, Wuhan, China), anti-β-actin polyclonal antibody (diluted 1:5000, Bioworld Technology Co., Ltd., MN), anti-HSP90B1 polyclonal antibody (diluted 1:1000, Elabscience), anti-albumin polyclonal antibody (diluted 1:1000, Abclonal, Boston, MA), anti-calreticulin polyclonal antibody (diluted 1:1000, Abways, Shanghai, China), anti-fatty acid binding protein 5 polyclonal antibody (diluted 1:500, Proteintech), anti-peroxiredoxin VI monoclonal antibody (diluted 1:2000, Abfrontier, Santiago, CA), anti-mouse-IgG antibody (diluted 1:5000, Bioworld) and anti-rabbit-IgG antibody (diluted 1:5000, Bioworld).

Techniques: Transgenic Assay, Western Blot