Journal: Nature Communications

Article Title: Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

doi: 10.1038/s41467-021-26830-7

Figure Lengend Snippet: a – e 1-week-old normal dogs were either co-injected with AAV.CK8.SpCas9 and AAV.RSV.AP or injected with AAV.RSV.AP only. a Representative HE, AP, CD4, and CD8 stainings. b Cas9 , and AP vector genome quantification (N, n = 5; others, n = 3). c Serum Cas9 antibody (Naive, n = 8; 6wks., n = 1). d Cas9 ELISpot assay on lymphocytes (Naive, n = 5; 6wks., n = 3). e Muscle cytokine transcript quantification (Naive, n = 8; others, n = 3). f - j Similar to a - e except 1-m-old dogs were injected. f Representative HE, AP, CD4, and CD8 stainings. Arrowhead, T-cell infiltration wiped out AP expression. g Cas9 and AP vector genome quantification ( N , n = 4; others, n = 3). h Serum Cas9 antibody ( N , n = 12; 6wks., n = 1). i Cas9 ELISpot assay on lymphocytes (Naive, n = 5; 6wks., n = 1). j Muscle cytokine transcript quantification ( N , n = 14; others, n = 3). k – p Similar to a - e except adult dogs were injected with either AAV.CK8.SpCas9 or AAV.RSV.AP. k Representative HE, AP, CD8, and granzyme B stainings. The boxed region was magnified. Yellow arrowhead, a dying myofiber lost dystrophin expression and infiltrated with CD8 + and granzyme B + T cells. l CD8 + T-cell quantification ( n = 3 for all categories). m Dystrophin and Cas9 western blot. n Serum Cas9 antibody (Naive, n = 27; others, n = 3). o Cas9 ELISpot assay on lymphocytes (Naive, n = 6; others, n = 3). p Muscle cytokine transcript quantification from adult dogs ( N , n = 12; others, n = 3). Immune cells are stained in dark brown, and AP is stained in blue. AP, alkaline phosphatase. N, No AAV. N/A, non-applicable. wks., weeks post-injection. Data are mean ± SEM. Statistical analysis was performed using Crawford-Howell test for c , h , i , One-way ANOVA with Tukey’s multiple comparisons for b , e , g , j , l , and n - p and Student’s t-test for d . See source data file for the exact p -value. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rat monoclonal IgG1 antibody against canine CD8 (Catalog# MCA1039, clone YCATE 55.9, Lot # 149349, 1:200, Bio-Rad Laboratories, Inc.) was used to detect CD8 + T and was visualized using a biotin-conjugated goat anti-rat IgG (H + L) antibody (A10517, Lot # 1441195, 1:1000, ThermoFisher Scientific).

Techniques: Injection, Plasmid Preparation, Enzyme-linked Immunospot, Expressing, Western Blot, Staining

Journal: Nature Communications

Article Title: Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

doi: 10.1038/s41467-021-26830-7

Figure Lengend Snippet: a Representative HE, dystrophin, CD4, and CD8 stainings from biopsy. b , Representative dystrophin and CD8 stainings from necropsied muscles. c Representative HE, dystrophin, CD8, and granzyme B staining from a necropsied muscle. d Cas9 and gRNA vector genome quantification (N, n = 7; 3wks., n = 1; 6wks., n = 11). e Cas9 transcript quantification ( N , n = 7; 3wks., n = 1; 6wks., n = 11). f Serum Cas9 antibody ( N , n = 16, 8, 9, 11, 12, 23, 11 and 18 for 0, 1, 2, 3, 4, 6, 8, and 10 weeks after birth, respectively; dog#1, n = 1 for 0, 4–10 weeks after birth). g PBMC IFN-γ ELISPOT against Cas9 ( N , n = 11; dog #1 n = 1 for all-time points). h Representative HE, dystrophin, CD4, and CD8 staining from biopsy. i Representative dystrophin and CD8 staining from necropsied muscles. j Representative HE, dystrophin, CD8, and granzyme B stainings from a necropsied muscle. k Cas9 and gRNA vector genome quantification ( N , n = 7; 3wks., n = 1; 6wks., n = 18). l Cas9 transcript quantification ( N , n = 7, 3wks., n = 1; 6wks., n = 18). m Serum Cas9 antibody ( N , n = 16, 8, 9, 11, 12, 23, 11 and 18 for 0, 1, 2, 3, 4, 6, 8, and 16 weeks after birth, respectively; dog#2, n = 1 for 0, 4–16 weeks after birth). n Cas9 specific IFN-γ ELISpot assay on PBMCs ( N , n = 11; dog #1 n = 1 for all-time points). o Representative HE, dystrophin, CD4 and CD8 staining from the control dog that received the micro-dystrophin vector. p Muscle cytokine transcript quantification from all three dogs ( N , n = 7;dog#1, 3wks. n = 1, 6wks. n = 11; dog#2, 3wks. n = 1, 12wks. n = 18; μDys injected dog, 6wks. n = 1, 24wks. n = 1). Immune cells are stained in dark brown. Arrow, a granzyme B-positive T cell. N , No AAV. wks., weeks post-injection. Data are mean ± SEM. Statistical analysis was performed using Crawford–Howell test for d – g and k – n , One-way ANOVA with Tukey’s multiple comparisons for p . See source data file for the exact p -value. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rat monoclonal IgG1 antibody against canine CD8 (Catalog# MCA1039, clone YCATE 55.9, Lot # 149349, 1:200, Bio-Rad Laboratories, Inc.) was used to detect CD8 + T and was visualized using a biotin-conjugated goat anti-rat IgG (H + L) antibody (A10517, Lot # 1441195, 1:1000, ThermoFisher Scientific).

Techniques: Muscles, Staining, Plasmid Preparation, Enzyme-linked Immunospot, Control, Injection

Journal: Nature Communications

Article Title: Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

doi: 10.1038/s41467-021-26830-7

Figure Lengend Snippet: a Representative HE, AP CD4, and CD8 stainings. b Cas9 and AP vector genome quantification ( N , n = 7; others, n = 1). c Cas9 and AP transcript quantification ( N , n = 7; others, n = 1). d AP + myofiber quantification ( n = 1 in all categories). e Serum Cas9 antibody ( N , n = 16, 8, 9, 11, 12, 23, 11 and 18 for 0, 1, 2, 3, 4, 6, 8, and 16 weeks after birth, respectively; dog#1, n = 1 for all-time points). f Cas9-specific IFN-γ ELISpot assay on PBMCs ( N , n = 11; dog #1 n = 1 for all-time points). g Representative HE, AP, CD4, and CD8 staining. h Cas9 and AP vector genome quantification ( N , n = 7; others, n = 1). i Cas9 and AP transcript quantification (in both panels: N , n = 7; each other group, n = 1). j AP + myofiber quantification ( n = 1 in each group). k Serum Cas9 antibody ( N , n = 16, 8, 9, 11, 12, 23, 11 and 18 for 0, 1, 2, 3, 4, 6, 8, and 16 weeks after birth, respectively; dog#2, n = 1 for all time points). l , PBMC IFN-γ ELISpot against Cas9 ( N , n = 11; dog #1 n = 1 for all time points). m Representative HE, AP, CD4, and CD8 stainings. n AP vector genome quantification ( N , n = 7; others, n = 1). o AP transcript quantification ( N , n = 7; others, n = 1). p Muscle cytokine transcript quantification from all 3 dogs at the indicated time points post-injection ( N , n = 14; dog#1, 3wks. n = 1, 6wks. n = 1, 12wks. n = 1; dog#2, 3wks. n = 1, 6wks. n = 1, 12wks. n = 1; dog injected with AP vector only, 6wks. n = 1, 52 wks. n = 1). AP, alkaline phosphatase. N, No AAV. wks., weeks post-injection. Data are mean ± SEM. Statistical analysis was performed using Crawford-Howell test for b , c , e , f , h , i , k , l , n , o , and One-way ANOVA with Tukey’s multiple comparisons for p . See source data file for exact p -value. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rat monoclonal IgG1 antibody against canine CD8 (Catalog# MCA1039, clone YCATE 55.9, Lot # 149349, 1:200, Bio-Rad Laboratories, Inc.) was used to detect CD8 + T and was visualized using a biotin-conjugated goat anti-rat IgG (H + L) antibody (A10517, Lot # 1441195, 1:1000, ThermoFisher Scientific).

Techniques: Plasmid Preparation, Enzyme-linked Immunospot, Staining, Injection

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Characterization of DOCK8 expression in horse lymphocytes. (A – C) Representative DOCK8 expression in different lymphocyte subsets. Flow cytometry analyses demonstrate that DOCK8 is expressed in CD4 + T cells, CD8 + T cells and in B cells (white histograms) with high intensity. White histograms represent specific stainings, grey histograms represent respective isotype controls. Y-axis show the amount of cells included in measurement; x-axis show the intensity of DOCK8 staining. (D) Statistical analyses of geo mean fluorescence intensity of DOCK8 expression in lymphocyte subsets of 12 healthy controls. While there was no difference between DOCK8 expression intensity in CD4 + T cells (white column) and CD8 + T cells (light grey column), DOCK8 was significantly higher (*p ≤ 0.05) expressed in B cells (dark grey column) compared to T cells.

Article Snippet: Staining of 5 × 10 5 cells per well was performed with anti-equine CD4 (mouse IgG1 monoclonal, Bio-Rad AbD Serotec, Puchheim, Germany; 1:100), PE-conjugated anti-equine CD8 (mouse IgG 2a monoclonal, Bio-Rad AbD Serotec; 1:10) and anti-equine B cells (clone CVS 41, monoclonal mouse IgG anti-horse Ig light chain, Bio-Rad AbD Serotec; 1:10) antibodies, diluted in FACS staining buffer (1% BSA + 0,001% NaN 3 in PBS).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Diminished DOCK8 protein levels in PBL of ERU cases. (A) Representative signal abundances of DOCK8 in PBL of controls (left panel) and ERU (right panel) detected by western blot. DOCK8 signal abundance in PBL of diseased specimen was decreased compared to controls. Lower molecular extra bands result from further reactivities of the polyclonal rabbit anti DOCK8 antibody. (B) Compared to healthy controls (n = 16), DOCK8 levels in ERU PBL (n = 36) were significantly (***p ≤ 0.001) decreased to 63% of physiological expression rate (set to a 100%). All protein abundances of DOCK8 were normalized to beta actin. (C) Analyzes of lymphocyte subset composition in healthy controls and autoimmune cases revealed no significant differences. (D) Statistical analysis of DOCK8 expression differences in lymphocyte subsets of healthy controls (white bars; n = 13) and ERU cases (grey bars; n = 12). DOCK8 expression intensity (geo MFI) was decreased in all lymphocyte subsets of ERU cases. In CD8 + T cells, no considerable DOCK8 expression difference between control PBL and ERU cases could be detected, whereas in CD4 + T cells of ERU horses, geo MFI was reduced to 85% compared to healthy controls. In B cells of autoimmune cases, a significant decrease of DOCK8 geo MFI to 57% was detected (*p ≤ 0.05).

Article Snippet: Staining of 5 × 10 5 cells per well was performed with anti-equine CD4 (mouse IgG1 monoclonal, Bio-Rad AbD Serotec, Puchheim, Germany; 1:100), PE-conjugated anti-equine CD8 (mouse IgG 2a monoclonal, Bio-Rad AbD Serotec; 1:10) and anti-equine B cells (clone CVS 41, monoclonal mouse IgG anti-horse Ig light chain, Bio-Rad AbD Serotec; 1:10) antibodies, diluted in FACS staining buffer (1% BSA + 0,001% NaN 3 in PBS).

Techniques: Western Blot, Expressing, Control