Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

doi: 10.1016/j.omtm.2019.01.003

Figure Lengend Snippet: Recombinant HBoV1 Vector Production and Analysis of Packaging Capacity (A) Plasmids for rAAV/HBoV1 production using either the 4-plasmid (constructs 1–4) or 3-plasmid (constructs 1, 2+3, and 4) transfection protocols. In both systems, the plasmids are co-transfected into HEK293T cells, which are then harvested 72 h later. To release the viral particles, cells are subjected to five freeze-thaw cycles, before free plasmid DNA is digested with benzonase. The resulting crude lysate is purified using iodixanol gradient centrifugation, and the vector-containing 40% phase is collected. (B) Purification of scAAV-YFP/HBoV1 via iodixanol gradient centrifugation. Shown is the distribution of benzonase-resistant particles in the different indicated iodixanol fractions. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan RT-PCR. (C) Production of scAAV-YFP/HBoV1 using the 3- or 4-plasmid transfection protocols. Data are mean (±SD) genome copies per milliliter (n = 3), as determined by TaqMan real-time PCR. (D) Oversized ssAAV-CRISPR constructs used in this work. Sp Cas9 and gRNA cassettes are expressed from different RNA polymerase II (Pol II) (first column) or Pol III (second column) promoters, respectively. Total genome sizes are shown in the third column. (E) Southern blot analysis of the ssAAV-CRISPR genomes from (D), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were detected with a probe against Sp Cas9. (F) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. (G) Oversized scAAV genomes used in this work. Stuffer sequences from lacZ with the indicated lengths (first column) were inserted to increase the total genome size (second column). (H) Southern blot analysis of the scAAV-YFP genomes from (G), which were packaged into and isolated from HBoV1 particles and then resolved on an alkaline agarose gel. The number above each lane indicates the size of the packaged genome. AAV vector genomes were labeled with a probe against yfp . (I) Low-molecular-weight (Hirt) extracts of the indicated constructs followed by Southern blot analysis. Brackets indicate monomeric (M) and dimeric (D) AAV replicative forms. ss, single-stranded; sc, self-complementary.

Article Snippet: This variant was now replaced with the Sp Cas9 cDNA from the Zhang lab, by PCR-amplifying this cDNA from former Addgene plasmid 49535 (in the meantime replaced by Addgene by plasmid 52961) using primers 46 and 47 and by cloning the NheI/ClaI-digested PCR product into our appropriately cleaved original AAV/ Sp Cas9 plasmid, resulting in pAAV-FZ Sp Cas9.

Techniques: Recombinant, Plasmid Preparation, Construct, Transfection, Purification, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, CRISPR, Southern Blot, Isolation, Agarose Gel Electrophoresis, Molecular Weight, Labeling

Journal: bioRxiv

Article Title: Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

doi: 10.1101/2025.02.14.637261

Figure Lengend Snippet: (A) Four guide sequences (sgKO.1, sgKO.2, sgKO.3, and sgKO.4) were designed to recognize exon 2 of the human ATXN3 gene (sgRNA target sequences are displayed in green). Sp Cas9 is recruited to the locus of interest, mediating the insertion of a DSB (colored scissors in the scheme and arrowheads in the target sequences) at approximately 3 base pairs upstream of a PAM sequence (sequence displayed in magenta). Subsequently, genome editing is achieved via NHEJ repair pathway for the permanent blocking of ATXN3 gene expression. (B) sgRNA sequences were cloned into a lentiviral expression vector (lentiCRISPRv2, addgene plasmid #52961), which also codifies for a FLAG-tagged Sp Cas9 and a puromycin resistance cassette. For the validation of the guide sequences, HEK293T cells were transfected with each of the generated plasmids and maintained in culture for 72 hours (selection medium with puromycin 10 µg/mL for 48 hours). Cells transfected with a guide sequence targeting the bacterial lacZ gene (sgCTRL) were used as a negative control. (C-D) Locus modification efficiencies were analyzed using Surveyor nuclease assay. Lane 1: DNA ladder (GeneRuler 100 bp, Thermo Fisher Scientific); Lane 2: Cells transfected with the sgCTRL construct; Lanes 3-6: Cells transfected with the sgRNA knock-out guide sequences (sgKO.1, sgKO.2, sgKO.3 and sgKO.4, respectively). Arrowheads indicate the expected DNA fragments, cleaved by Surveyor nuclease. The estimated indel occurrence within human ATXN3 locus is represented as a percentage (n=4). (E-F) Western blot analysis revealed a significant decrease (approximately 0.5-fold change) in ATXN3 protein levels after Sp Cas9 targeting of ATXN3 locus in comparison with the control sequence (n=3). Results are presented as fold change relative to cells transfected with the sgCTRL expressing plasmid. Optical densitometry analysis of ATXN3 fractions were normalized with β-actin and FLAG signals. Statistical significance was evaluated with one-way ANOVA with Dunnett’s post hoc test (*p<0.05). Data are expressed as mean ± SEM. Abbreviations : LTR (long terminal repeat); U6 (Pol III promoter); sgRNA (single guide RNA); EFS (elongation factor 1α short promoter); Sp Cas9 (Cas9 nuclease from Streptococcus pyogenes) ; NLS (nuclear localization signal); FLAG (FLAG octapeptide tag); P2A (2A self-cleaving peptide); Puro (puromycin selection marker); WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Article Snippet: Due to constraints related with AAV packaging capacity, a two-vector system was adopted for in vivo applications : i) AAV- Sp Cas9 (vector pX551, plasmid #60957, Addgene) and ii) AAV- Sp Guide (vector pX552, plasmid #60958, Addgene).

Techniques: Sequencing, Blocking Assay, Expressing, Clone Assay, Plasmid Preparation, Transfection, Generated, Selection, Negative Control, Modification, Nuclease Assay, Construct, Knock-Out, Western Blot, Comparison, Control, FLAG-tag, Marker, Virus

Journal: bioRxiv

Article Title: Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

doi: 10.1101/2025.02.14.637261

Figure Lengend Snippet: (A-B) Schematic representation of the stereotaxic co-injection of viral vectors in the striatum of C57BL/6 mice. Lentivirus encoding for the human mutant ATXN3 protein with 72Q (Myc-tagged), rAAV1/2 encoding for Sp Cas9 (HA-tagged) and rAAV1/2 encoding for the CTRL guide sequence (EGFP-KASH co-expression) were injected in the left hemisphere, serving as experimental control. In the contralateral hemisphere rAAV1/2 encoding for the sgKO.2 sequence were injected, along with LV-PGK- ATXN3 72Q and the rAAV1/2- Sp Cas9. Four weeks after surgery animals were sacrificed. (C) Western blot analysis of striatal homogenates demonstrates that CRISPR- ATXN3 KO system promotes a reduction of mutant ATXN3 species in treated hemispheres, when compared with the contralateral control hemispheres (data not quantified). (D-E) Immunohistochemical peroxidase staining upon labelling of striatal sections with anti-ubiquitin antibody, 4 weeks after stereotaxic surgery. Scale bar, 50 µm. (F) CRISPR- ATXN3 KO injected hemispheres display a drastic reduction in the number of ubiquitin-positive inclusions in comparison with the contralateral control hemisphere, injected with CRISPR-CTRL. (G-H) Immunohistochemical analysis using anti-DARPP-32 antibody for expanded ATXN3-derived lesion identification. Treated hemispheres, injected with CRISPR- ATXN3 KO showed a statistically significant reduction of DARPP-32 depleted volume, as quantified in (I) . Scale bar, 200 µm. (J-K) Iba-1 immunoreactivity in mouse striata. No statistically significant differences are observed between control (J) and CRISPR-edited hemispheres (K), as quantified in (L) . Scale bar, 200 µm in general view images and 50 µm in detail magnifications. (M-N) Gfap immunoreactivity in mouse striata. No statistically significant differences in the Gfap immunoreactivity are observed between non-edited (M) and CRISPR-edited striata (N), as quantified in (O) . Scale bar, 200 µm in general view images and 50 µm in detail magnifications. Statistical significance was evaluated with paired Student’s t-test (**p<0.01, n=5). Data are expressed as mean ± SEM. Abbreviations : ITR (invert terminal repeat); pMecp2 (mouse methyl CpG binding protein 2 promoter); HA (hemagglutinin tag); NLS (nuclear localization signal); Sp Cas9 (Cas9 nuclease from Streptococcus pyogenes) ; U6 (Pol III promoter); sgRNA (single guide RNA); hSyn1 (human synapsin 1 promoter); EGFP (enhanced green fluorescent protein); KASH (Klarsicht, ANC1, Syne Homology nuclear transmembrane domain); hGHpA (human growth hormone gene polyadenylation signal).

Article Snippet: Due to constraints related with AAV packaging capacity, a two-vector system was adopted for in vivo applications : i) AAV- Sp Cas9 (vector pX551, plasmid #60957, Addgene) and ii) AAV- Sp Guide (vector pX552, plasmid #60958, Addgene).

Techniques: Injection, Mutagenesis, Sequencing, Expressing, Control, Western Blot, CRISPR, Immunohistochemical staining, Staining, Comparison, Derivative Assay, Binding Assay