erythroleukemic cell line  (ATCC)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Ligand-modified rAAV6 vectors with nanoblades allow high-level gene knockin in HSPCs without compromising cell survival

doi: 10.1016/j.omtn.2025.102495

Figure Lengend Snippet: Coupling of rAAV6 with low equivalents of mannose increased transduction of CD34 + cells while preventing induction of cell death (A and B) K562 cells were transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (control [Ctrl]) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5; see also Table S1 ). At day 3 and 6 post transduction, the % of GFP-positive K562 cells (A) and their survival (B) were determined by FACS. Mean (SD); n = 3 biological repeats; multiple Student’s t test, Tukey’s multiple comparisons test, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001. (C and D) CD34 + cells were pre-stimulated with cytokines overnight and were then transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (Ctrl) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5). At day 3 and 6 post transduction, the % of GFP-positive CD34 + cells (C) and their survival using DAPI staining (D) was determined by FACS. Mean (SD); n = 3; three biological repeats, for which three independent CD34 + cell donors were used; multiple Student’s t test, Tukey’s multiple comparisons test; ∗ p < 0.05,∗∗∗∗ p < 0.0001.

Article Snippet: The human erythroleukemic cell line K562 (ATCC, Manassas, VA; CCL-243) and the Raji cell line (ATCC; Manassas, VA; CCL-86) were maintained in RPMI 1640 medium (Gibco-BRL, Middlesex, UK), supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Transduction, Control, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Ligand-modified rAAV6 vectors with nanoblades allow high-level gene knockin in HSPCs without compromising cell survival

doi: 10.1016/j.omtn.2025.102495

Figure Lengend Snippet: Bio-conjugation of rAAV6 with mannose allowed high-level nanoblade-mediated knockin into CD34 + cells and reduced cell death (A and B) K562 cells were transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (Ctrl) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5) in the presence of nanoblades. At day 3 and 6 post incubation with rAAV vectors and nanoblades, the % of knockin was determined by % of (A) GFP-expressing K562 cells and their survival using DAPI staining (B) analyzed by FACS; mean (SD); n = 3; three biological repeats; multiple Student’s t test, Tukey’s multiple comparisons test; ∗∗∗∗ p < 0.001. (C and D) CD34 + cells were pre-stimulated with cytokines overnight and were then transduced at an MOI of 50,000 (vg/cell) with the unmodified rAAV2 and rAAV6 vectors (Ctrl) or with rAAV2 and rAAV6 bio-conjugated with mannose sugars at their lysine residues (K3E5 or K3E6) or tyrosine residues (Y3E5; see also Table S1 ) in the presence of nanoblades. At day 3 and 6 post transduction, the % of GFP-positive CD34 + cells (% knockin; C) and their survival (D) were determined using FACS analysis; mean (SD); n = 3; three biological repeats for which three independent CD34 + cell donors were used; multiple Student’s t test, Tukey’s multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (E) Representative FACS analysis for the data in (C) showing gene knockin by analysis of GFP expression in the CD34 + cells at day 6.

Article Snippet: The human erythroleukemic cell line K562 (ATCC, Manassas, VA; CCL-243) and the Raji cell line (ATCC; Manassas, VA; CCL-86) were maintained in RPMI 1640 medium (Gibco-BRL, Middlesex, UK), supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Conjugation Assay, Knock-In, Incubation, Expressing, Staining, Transduction