Journal: Journal of Mother and Child

Article Title: Gene Therapy for Inherited Metabolic Diseases

doi: 10.34763/jmotherandchild.20202402si.2004.000009

Figure Lengend Snippet: Indicative list of gene therapy clinical trials for inherited metabolic diseases in 2020.

Article Snippet: , , Sangamo Therapeutics , I/II , R , AAV6 , NCT04046224.

Techniques: Clinical Proteomics, Plasmid Preparation

Journal: BMC Genomics

Article Title: Cas9 -expressing cattle using the PiggyBac transposon all-in-one system

doi: 10.1186/s12864-025-11381-8

Figure Lengend Snippet: Gene engineering using Cas9 -expressing germ cells via viral and non-viral methods. A Schematic experiment design for testing sgRNA for PRNP gene knockout utilizing viral and nonviral approaches without introducing Cas9 during the pre-embryonic in vitro culture process. B An SB transposon vector containing both GFP and sg PRNP sequences was microinjected, resulting in mutations identified in embryos that expressed both RFP and GFP (yellow arrow: R + and G + , white arrow: R- and G +). C T7E1 assay result confirming PRNP mutations (Lanes: M: marker, WT: wild type, MI: microinjection, R + : RFP positive, G + : GFP positive, R-: RFP negative, NC: negative control, and PC: positive control). D and E The viral method for gene editing was carried out using AAV6 containing an sg PRNP sequence. T7E1 assay results of PRNP mutation in the RFP -positive group cultured for 72 h after media treatment with AAV6. F Schematic experiment design for knock-in strategy at the BSA gene locus in embryos. G Observation of GFP -positive blastocysts post-AAV6 infection. H Schematic of the knock-in strategy and validation. Diagram showing targeting site on Bos taurus chromosome 6, where sgRNA targets bovine serum albumin (BSA). knock-in donor containing Attb-AfIII sequences for homologous recombination, flanked by 500-bp left and right homology arms (LHA and RHA) (a). PCR validation of the knock-in band. Eight randomly selected GFP -positive blastocysts were sampled, and PCR was carried out with primers targeting sequences both inside and outside the knock-in regions (b). I Summary table of the knock-in efficiency (results from three experiments) including number of cumulus-oocyte complexes (COCs), 8-cell stage embryos, total blastocysts (BL), GFP -positive blastocysts ( GFP + BL), and percentage of knock-in in sampled blastocysts

Article Snippet: This AAV6 was specifically designed and produced by company (Vigene Biosciences, US).

Techniques: Expressing, Gene Knockout, In Vitro, Plasmid Preparation, Marker, Microinjection, Negative Control, Positive Control, Sequencing, Mutagenesis, Cell Culture, Knock-In, Infection, Biomarker Discovery, Homologous Recombination

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: 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: 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: 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