Journal: Molecular Therapy

Article Title: Generation of a Kupffer Cell-evading Adenovirus for Systemic and Liver-directed Gene Transfer

doi: 10.1038/mt.2011.71

Figure Lengend Snippet: Ad5/6GL is taken up into macrophages less than unmodified Ad5GL. (a) adenovirus (Ad) vectors were added to RAW 267.4 cells (multiplicity of infection 10,000). Cells were harvested 24 hours later and genomic DNA was extracted. Real-time PCR was used to quantify viral and cellular genomes using cytomegalovirus (CMV) and GAPDH specific primers, respectively. Data are normalized to cellular genomes. N = 4. BALB/c mice were predosed intravenous (i.v.) with 3 × 1010 virus particles (vp) in 100 µl in phosphate-buffered saline (PBS). After 4 hours, the mice were injected with 1 × 1010 vp in PBS of unmodified or chimeric Ad vector. Mice were imaged and luciferase expression was quantified (b) 24 hours after Ad vector injection and (c) over time. N = 5. (d) Varying doses of Ad vectors were injected i.v. into mice. After 1 hour, mice were euthanized and 20 ng of tissue from the large liver lobe was harvested. Total DNA was extracted and real-time PCR was used to quantify viral genomes using CMV primers. *P < 0.05; **P < 0.005; ns = not significant. Means ± SEM.

Article Snippet: Ad5/6GL: Ad6 viral genomic DNA was purified using a PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA) and the HVR cassette was amplified by PCR (F primer 5′-TACTTTGACATCCGCGGCGTGCTGG-3′ and R primer 5′-GTGGGGCCATGGGGAAGAAGGTGGCG-3′), cut with Apa I and Pst I and cloned into the Ad5GL backbone.

Techniques: Infection, Real-time Polymerase Chain Reaction, Virus, Saline, Injection, Plasmid Preparation, Luciferase, Expressing

Journal: Molecular Therapy

Article Title: Generation of a Kupffer Cell-evading Adenovirus for Systemic and Liver-directed Gene Transfer

doi: 10.1038/mt.2011.71

Figure Lengend Snippet: Expression of hFIX from intravenous (i.v.) injection of helper dependent adenovirus (Ad) vectors. Helper dependent (HD) vectors were injected intravenously into BALB/c mice at 2.5 × 1010 virus particles (vp)/mouse. Serum was harvested on (a) day 7 and (b) up to day 21. Serum was analyzed for human Factor IX by enzyme linked imunosorbent assay. N = 5 for Ad5 and Ad5/6; N = 4 for Ad6. **P < 0.005; ns = not significant. Means ± SEM.

Article Snippet: Ad5/6GL: Ad6 viral genomic DNA was purified using a PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA) and the HVR cassette was amplified by PCR (F primer 5′-TACTTTGACATCCGCGGCGTGCTGG-3′ and R primer 5′-GTGGGGCCATGGGGAAGAAGGTGGCG-3′), cut with Apa I and Pst I and cloned into the Ad5GL backbone.

Techniques: Expressing, Injection, Virus

Journal: Gene Therapy

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9

doi: 10.1038/s41434-018-0003-1

Figure Lengend Snippet: Adenoviral-mediated delivery of CRISPR/Cas9 for targeted gene editing. Genetic designs of a CRISPR/Cas9-based strategy for targeted homologous recombination. Ad5-Cas9-gRNA ( a ) expresses both the Cas9 gene and a ROSA26- targeting gRNA sequence from the E1- and the E3-deleted regions, respectively. Ad5-Cas9 ( b ) expresses the Cas9 gene from the E1-deleted region. Ad5-gRNA ( c ) contains an E1-deleted region and expresses the ROSA26 gRNA from the E3-deleted region. Ad5-EF1α-EGFP ( d ) expresses the EGFP gene from the mammalian EF1α promoter/intron and contains 0.8 kb length homology arms to the ROSA26 locus surrounding the expression cassette, in the E1-deleted region. Ad5-EF1α-hAAT ( e ) expresses the hAAT gene from the same expression cassette and homology arms as the EGFP donor vector

Article Snippet: Fig. 2 Targeted in vitro knock-in occurs following adenoviral delivery of CRISPR/Cas9. a Targeted Illumina deep sequencing of the ROSA26 locus was performed on genomic DNA extracted from the BNL-1NG murine liver cell line following transduction with Ad5-Cas9-gRNA infection at various amounts of viral particles per cell (VP/cell), to determine insertion and deletion (indel) formation resulting from NHEJ DNA repair of DSBs. b BNL-1NG cells were transduced with either Ad5-EF1α-EGFP (donor) and Ad5-Cas9 (no gRNA) or Ad5.EF1α-EGFP (donor) and Ad5-Cas9-gRNA.

Techniques: CRISPR, Homologous Recombination, Sequencing, Expressing, Plasmid Preparation

Journal: Gene Therapy

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9

doi: 10.1038/s41434-018-0003-1

Figure Lengend Snippet: Targeted in vitro knock-in occurs following adenoviral delivery of CRISPR/Cas9. a Targeted Illumina deep sequencing of the ROSA26 locus was performed on genomic DNA extracted from the BNL-1NG murine liver cell line following transduction with Ad5-Cas9-gRNA infection at various amounts of viral particles per cell (VP/cell), to determine insertion and deletion (indel) formation resulting from NHEJ DNA repair of DSBs. b BNL-1NG cells were transduced with either Ad5-EF1α-EGFP (donor) and Ad5-Cas9 (no gRNA) or Ad5.EF1α-EGFP (donor) and Ad5-Cas9-gRNA. Additional controls consisted of no virus and a sham Ad5 virus expressing an irrelevant coagulation factor gene driven by the CMV gene and lacking any CRISPR/Cas9 components. Genomic DNA was extracted for PCR amplification of integration junctions and 612 bp amplicon of viral hexon gene as a loading control to confirm the presence of viral vector transduction. Primer sets are composed of one primer within the genome (outside of the homology arms) and one primer within the donor transgene, to confirm on-target insertions. Junction PCR products were gel extracted and Sanger sequenced to confirm targeted integration (Supplemental Figs. and ). Gels are representative of four independent experiments with junction amplification being confirmed from early and late time points. Arrows show direction and position of PCR primers

Article Snippet: Fig. 2 Targeted in vitro knock-in occurs following adenoviral delivery of CRISPR/Cas9. a Targeted Illumina deep sequencing of the ROSA26 locus was performed on genomic DNA extracted from the BNL-1NG murine liver cell line following transduction with Ad5-Cas9-gRNA infection at various amounts of viral particles per cell (VP/cell), to determine insertion and deletion (indel) formation resulting from NHEJ DNA repair of DSBs. b BNL-1NG cells were transduced with either Ad5-EF1α-EGFP (donor) and Ad5-Cas9 (no gRNA) or Ad5.EF1α-EGFP (donor) and Ad5-Cas9-gRNA.

Techniques: In Vitro, Knock-In, CRISPR, Sequencing, Transduction, Infection, Virus, Expressing, Coagulation, Amplification, Control, Plasmid Preparation

Journal: Gene Therapy

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9

doi: 10.1038/s41434-018-0003-1

Figure Lengend Snippet: Knock-in and NHEJ rates following in vivo delivery with adenovirus is variable and persists over time. A total of 1 × 10 11 viral particles of Ad5-EF1α-EGFP and Ad5-Cas9-gRNA ( n = 6), or Ad5-EF1α-EGFP and sham Ad5-gRNA ( n = 6), were injected into mice at a ratio of 1:1. PBS mock injected mice served as a no virus control ( n = 3). a Junction capture PCR was used to amplify 3′ junctions from EGFP- integrated ROSA26 alleles from genomic DNA from the mice. Each lane represents genomic DNA template from one mouse. Viral amplification of 612 bp of the hexon gene served as a loading control and confirmed viral transduction. b LAM-PCR was performed by production of single-stranded linear PCR product from a biotinlyated primer binding downstream of the 3′ homology region of ROSA26 in the mouse genome, whereby both integrated and non-integrated alleles are amplified. qPCR analysis to quantify HDR-mediated integration rates is displayed as percent EGFP- integrated alleles out of total ROSA26 alleles. Error bars are s.d. of the mean. c Mice injected with Ad5-Cas9-gRNA or a sham virus were sacrificed at time points ranging between 7 and 210 days and genomic DNA was extracted. This DNA, and genomic DNA extracted at the conclusion of the hAAT experiment in Figs. and , was used as template for targeted Illumina deep sequencing of the ROSA26 for the presence of edited alleles, witnessed by NHEJ-induced indels

Article Snippet: Fig. 2 Targeted in vitro knock-in occurs following adenoviral delivery of CRISPR/Cas9. a Targeted Illumina deep sequencing of the ROSA26 locus was performed on genomic DNA extracted from the BNL-1NG murine liver cell line following transduction with Ad5-Cas9-gRNA infection at various amounts of viral particles per cell (VP/cell), to determine insertion and deletion (indel) formation resulting from NHEJ DNA repair of DSBs. b BNL-1NG cells were transduced with either Ad5-EF1α-EGFP (donor) and Ad5-Cas9 (no gRNA) or Ad5.EF1α-EGFP (donor) and Ad5-Cas9-gRNA.

Techniques: Knock-In, In Vivo, Injection, Virus, Control, Amplification, Transduction, Binding Assay, Sequencing

Journal: Gene Therapy

Article Title: Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9

doi: 10.1038/s41434-018-0003-1

Figure Lengend Snippet: Off-target analysis and vector toxicity show little long-term consequences of gene knock-in. a Computationally derived top off-target candidate sites ( Syn3 , Ano6 , Nft3 , and chr5: 46645544) were submitted to targeted Illumina deep sequencing for evidence of NHEJ activity based upon indel formation following imperfect DNA repair. Amount of virus administered had no effect on the presence of indel formation at the selected candidate off-target sites. Each dot represents data from one individual mouse. b Mice were submitted in a blinded manner to gross pathology examination with an emphasis on liver toxicity markers (ALT/AST transaminases) and whole blood cell counts levels, 7 months after receiving hAAT-encoding vectors. Each dot represents data from one mouse. Error bars are s.d. of the mean. hAAT donor is Ad5-EF1α-hAAT and GFP sham is Ad5-CMV-EGFP. WBC white blood count, RBC red blood count, HGB hemoglobin, MCH mean cell hemoglobin

Article Snippet: Fig. 2 Targeted in vitro knock-in occurs following adenoviral delivery of CRISPR/Cas9. a Targeted Illumina deep sequencing of the ROSA26 locus was performed on genomic DNA extracted from the BNL-1NG murine liver cell line following transduction with Ad5-Cas9-gRNA infection at various amounts of viral particles per cell (VP/cell), to determine insertion and deletion (indel) formation resulting from NHEJ DNA repair of DSBs. b BNL-1NG cells were transduced with either Ad5-EF1α-EGFP (donor) and Ad5-Cas9 (no gRNA) or Ad5.EF1α-EGFP (donor) and Ad5-Cas9-gRNA.

Techniques: Plasmid Preparation, Gene Knock-In, Derivative Assay, Sequencing, Activity Assay, Virus