Journal: Stem Cell Research & Therapy

Article Title: Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

doi: 10.1186/s13287-021-02565-6

Figure Lengend Snippet: HDR gene-editing. Cas9-RNP along with a donor template (AAV6 or ssODN or IDLV) is delivered into HSPCs. Cas9 RNP introduces DNA double-stranded breaks at the target locus and endogenous HDR pathway repairs the DNA damage. During this process, the donor template having homologous sequence to the cut site is inserted into the target locus

Article Snippet: Gaucher disease , Cas9 , CCR5 , Cas9 RNP and AAV6 , Electroporation/transduction (Lonza 4D nucleofector) , HSPCs from healthy donor , [ ] .

Techniques: Sequencing

Journal: Stem Cell Research & Therapy

Article Title: Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

doi: 10.1186/s13287-021-02565-6

Figure Lengend Snippet: Therapeutic HDR gene-editing strategy for genetic disorders using ZFNs, TALENs and Cas9

Article Snippet: Gaucher disease , Cas9 , CCR5 , Cas9 RNP and AAV6 , Electroporation/transduction (Lonza 4D nucleofector) , HSPCs from healthy donor , [ ] .

Techniques: TALENs, Electroporation, Plasmid Preparation, Transfection, Transduction

Journal: Research

Article Title: AAV for Gene Therapy in Ocular Diseases: Progress and Prospects

doi: 10.34133/research.0291

Figure Lengend Snippet: Completed and ongoing gene replacement trials for ocular disease

Article Snippet: , RPE65 , SR , AAV6 , I/II , MeiraGTx UK II Ltd , 02946879.

Techniques: Plasmid Preparation

Journal: Nature Communications

Article Title: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

doi: 10.1038/s41467-021-24017-8

Figure Lengend Snippet: A Potential editing strategy for the generation of allogenic T cells. Multiplexed knock-out of TRAC , B2M , and CIITA to reduce the risk of host-versus-graft rejection and graft-versus-host disease responses, respectively. B Simultaneous triple knockout of TRAC , B2M , and CIITA (Class II Major Histocompatibility Complex Transactivator) by AsCas12a Ultra (1 µM RNP). The optimal guide RNA ( n = 1) targeting each locus was used for multiplex editing. Data are presented as mean values ± SD. C Workflow for generation of allogenic T cells with site-specific transgene knock-in by AsCas12a Ultra. The activated T cells were electroporated with Ultra RNPs and transduced with AAV6 carrying a donor template post-electroporation, then analyzed 3–4 days later for transgene expression by flow cytometry. D The knock-in efficiency of a fluorescent reporter at either TRAC or B2M locus using 1 µM RNP and 1 × 10 5 vg/cell AAV6. The optimal gRNA targeting each locus (n = 1) was used for this experiment. Alternative RNPs (Alt RNP, i.e., TRAC RNP + B2M AAV or B2M RNP + TRAC AAV) were used as negative control to rule out any non-specific integration of AAV donor. Data are presented as mean values ± SD. Paired, two-tailed t-test was performed to evaluate statistical significance (** P -value = 0.0012; *** P -value = 0.0008). E Simultaneous double knock-in of fluorescent reporters at TRAC and B2M across a range of RNP concentrations. The optimal gRNA targeting each locus ( n = 1) was used for this experiment. Raw source data are provided in the Source Data File.

Article Snippet: All AAV6 knock-in experiments followed the Lonza format procedures for RNP only experiments.

Techniques: Knock-Out, Triple Knockout, Immunopeptidomics, Multiplex Assay, Knock-In, Transduction, Electroporation, Expressing, Flow Cytometry, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

doi: 10.1038/s41467-021-24017-8

Figure Lengend Snippet: A Potential editing strategy for the generation of edited NK cells to overcome tumor microenvironment. Knocking out TGFBR2 (Transforming growth factor-beta receptor type 2) prevents inactivation by tumor-derived TGF-β1. B Editing efficiency of TGFBR2A across a range of RNP concentrations in NK cells derived from three different donors. Data are presented as mean values ± SD. C Tumor spheroid killing mediated by TGFBR2 knock-out NK cells. Engineered NK cells were co-cultured with SK-OV-3 ovarian tumor spheroids at effector-to-target cells ratio of 10:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (**** P -value ≤ 0.0001). Data are representative of five independent experiments using six unique NK cell donors. D Graphical depiction of EGFR CAR NK cell recognition of an EGFR + tumor cell. E Knock-in efficiency of a fluorescent reporter at either TRAC (Target site 1 and Target site 2) or B2M (Target site 3) loci using 4 μM RNP and 2.5 × 10 4 vg/cell AAV6 titer carrying the transgenes. F Tumor spheroid killing mediated by GFP + or αEGFR CAR + NK cells. Engineered NK cells were co-cultured with EGFR + PC-3 spheroids at effector-to-target cell ratios of 2:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (**** P -value ≤ 0.0001). Raw source data are provided in the Source Data File.

Article Snippet: All AAV6 knock-in experiments followed the Lonza format procedures for RNP only experiments.

Techniques: Derivative Assay, Knock-Out, Cell Culture, Cell Analysis, Knock-In

Journal: Nature Communications

Article Title: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

doi: 10.1038/s41467-021-24017-8

Figure Lengend Snippet: A Potential editing strategy for the generation of allogenic T cells. Multiplexed knock-out of TRAC , B2M , and CIITA to reduce the risk of host-versus-graft rejection and graft-versus-host disease responses, respectively. B Simultaneous triple knockout of TRAC , B2M , and CIITA (Class II Major Histocompatibility Complex Transactivator) by AsCas12a Ultra (1 µM RNP). The optimal guide RNA ( n = 1) targeting each locus was used for multiplex editing. Data are presented as mean values ± SD. C Workflow for generation of allogenic T cells with site-specific transgene knock-in by AsCas12a Ultra. The activated T cells were electroporated with Ultra RNPs and transduced with AAV6 carrying a donor template post-electroporation, then analyzed 3–4 days later for transgene expression by flow cytometry. D The knock-in efficiency of a fluorescent reporter at either TRAC or B2M locus using 1 µM RNP and 1 × 10 5 vg/cell AAV6. The optimal gRNA targeting each locus (n = 1) was used for this experiment. Alternative RNPs (Alt RNP, i.e., TRAC RNP + B2M AAV or B2M RNP + TRAC AAV) were used as negative control to rule out any non-specific integration of AAV donor. Data are presented as mean values ± SD. Paired, two-tailed t-test was performed to evaluate statistical significance (** P -value = 0.0012; *** P -value = 0.0008). E Simultaneous double knock-in of fluorescent reporters at TRAC and B2M across a range of RNP concentrations. The optimal gRNA targeting each locus ( n = 1) was used for this experiment. Raw source data are provided in the Source Data File.

Article Snippet: For knock-in experiments, T cells or NK cells were transduced with AAV6 (Sirion Biotech) containing the cargo of interest at the denoted MOI within 30 min after electroporation.

Techniques: Knock-Out, Triple Knockout, Immunopeptidomics, Multiplex Assay, Knock-In, Transduction, Electroporation, Expressing, Flow Cytometry, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

doi: 10.1038/s41467-021-24017-8

Figure Lengend Snippet: A Potential editing strategy for the generation of edited NK cells to overcome tumor microenvironment. Knocking out TGFBR2 (Transforming growth factor-beta receptor type 2) prevents inactivation by tumor-derived TGF-β1. B Editing efficiency of TGFBR2A across a range of RNP concentrations in NK cells derived from three different donors. Data are presented as mean values ± SD. C Tumor spheroid killing mediated by TGFBR2 knock-out NK cells. Engineered NK cells were co-cultured with SK-OV-3 ovarian tumor spheroids at effector-to-target cells ratio of 10:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (**** P -value ≤ 0.0001). Data are representative of five independent experiments using six unique NK cell donors. D Graphical depiction of EGFR CAR NK cell recognition of an EGFR + tumor cell. E Knock-in efficiency of a fluorescent reporter at either TRAC (Target site 1 and Target site 2) or B2M (Target site 3) loci using 4 μM RNP and 2.5 × 10 4 vg/cell AAV6 titer carrying the transgenes. F Tumor spheroid killing mediated by GFP + or αEGFR CAR + NK cells. Engineered NK cells were co-cultured with EGFR + PC-3 spheroids at effector-to-target cell ratios of 2:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (**** P -value ≤ 0.0001). Raw source data are provided in the Source Data File.

Article Snippet: For knock-in experiments, T cells or NK cells were transduced with AAV6 (Sirion Biotech) containing the cargo of interest at the denoted MOI within 30 min after electroporation.

Techniques: Derivative Assay, Knock-Out, Cell Culture, Cell Analysis, Knock-In

Journal: Cell host & microbe

Article Title: Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation

doi: 10.1016/j.chom.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AAV6 mCherry , Signagen , SL101273.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Inhibition, Protease Assay, Blocking Assay, Plasmid Preparation, Extraction, Western Blot, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: iPSC-hepatocyte organoids as a novel platform to predict AAV gene therapy efficacy

doi: 10.1016/j.omtm.2025.101467

Figure Lengend Snippet: Analysis of AAV transduction in cell lines (A) Phase-contrast and immunofluorescence images of AAV2 and AAV8 transduction in HepG2 and AAV6 and AAV8 transduction in HepaRG cell lines at day 1 and day 3. Scale bar, 200 μm. (B) AAV transduction efficiency overtime in percentage for HepG2 or HepaRG during 3 days. Data are expressed as mean (SD) ( n = 3). (C) Heatmap representing the mean of the percentage of AAV-transduced cells 3 days after AAV treatment, for HepG2 and HepaRG.

Article Snippet: AAV constructs produced in Sf9 cells through infection with two recombinant baculoviruses were sourced from Virovek: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, and AAV9 expressing green fluorescent protein (GFP) and empty AAV2, AAV8, and AAV9 were used under a cytomegalovirus (CMV) promoter.

Techniques: Transduction, Immunofluorescence