Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 1. Schematic representation of experimental protocol and workflow of this present work. The top panel is the schematic illustration of the SNA substrates inserts into commercial dishes for CD34+HSPCs enrichment. Below which is the schematic representation of SMNP delivery CBE- and sgRNA-plasmids into commercial dishes with SNA substrates inserts. Then, after obtaining umbilical cord blood cells, SNA substrate and SMNP delivery mediated CBE base editing (SNA⋅SMNP⋅CBE)— enable efficiently and precisely modify BCL11A promoter to achieve C-T conversion and HBG elevation in CD34+HSPCs. The edited human CD34+HSPC was successively transplanted into SCID mouse by intraosseous injection to detect the CD34 engraftment.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 3. supramolecular nanoparticle (SMNP) enables delivery of CBE and sgRNA plasmids to modify BCL11A to achieve HBG over expression in vitro. (A), Schematic representation of base-editing experiments in human umbilical cord blood derived CD34+HSPCs enriched by SNA substrate CBE-, sgRNA-(with a red fluorescent label (mCherry) expressing plasmid were co-delivered by SMNP in human CD34+HSPCs enriched by SNA substrate. (B), Representative images of mCherry + cells at 2 h post-transfection following SMNP delivery CBE-, sgRNA-plasmids. (C), Representative flow cytometry results of edited CD34 positive cells ratio after SMNP delivery CBE- and sgRNA- plasmids. (D), Editing efficiency of targeted BCL11A PCR products was detected by Sanger sequencing, with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2.(E), Real-time PCR analysis of mCherry (left panel), BCL11A (middle panel) and HBG (right panel) expression in CD34+HSPCs subjected with/without SMNP delivering co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids (edited CD34+HSPCs/ naïve CD34+HSPCs). CD34+HSPCs delivered by SMNP co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids was designated as edited group, and unedited group. Results are normalized to GAPDH and shown as mean±SEM. (n=3 biological replicates). Statistical tests compare edited samples and unedited control group (**P＜ 0.01). (F), Western blots were used to detect HBG expression at protein level in edited CD34+HSPCs and naïve CD34+HSPCs. Results are representative of three biological replicates.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Over Expression, In Vitro, Derivative Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 4. Comparison of our novel system and conventional strategy in the aspect of editing efficiency and CD34+HSPCs property. (A), Editing efficiency of targeted BCL11A PCR products from CD34+HSPCs subjected to different strategies. The top panel (SNA⋅SMNP group) is CD34+HSPCs was enriched by our SNA substrate and transfected with CBE- and sgRNA targeting BCL11A- plasmids by SMNP delivery (with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2). The middle panel (IMS⋅EP group) is CD34+HSPCs was isolated by conventional immunomagnetic bead method (IMS) and transfected CBE- and sgRNA targeting BCL11A- plasmids with electroporation (EP) (with C-T conversion value of 33.7 % base edits at position C1, and 10.2 % at position C2). The bottom panel (control group) was CD34+HSPCs without edited treatment. (B), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups: SNA⋅SMNP treated CD34+HSPCs, IMS⋅EP treated CD34+HSPCs and naïve CD34+ (right panel). (C), CD34 mRNA expression in three groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates). (D) mRNA expression of hematopoietic stem cell marker (CD133, CD90) and cell differentiation markers (GATA-1, SPI1, CD235a and CD33) in four groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs, naïve CD34+HSPCs, and PMBC. CD133 and CD90 were shown in left panel, while differ entiation markers including GATA-1, SPI1, CD235a and CD33 were presented in right panel. Results shown as mean±SEM (n=3 biological replicates).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, Transfection, Isolation, Electroporation, Control, Quantitative Proteomics, Expressing, Marker, Cell Differentiation

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 5. Tracks of editing feasibility of SNA to maintain CD34 property by Intraosseous injection of transplanted edited CD34+HSPCs into SCID mouse. (A), Gene edited human CD34+HSPCs were transplanted into SCD mouse with two different injection strategies: intraosseous injection into the bone marrow of the mouse tibia (left panel) and tail vein injection (right panel). (B), Representative immunofluorescence images (scale bar=8μm) of CD34 in the bone marrow (left panel) and peripheral blood of mice at 14 weeks of intraosseous injection (top) and tail vein injection (below) strategy, respectively. (C), Representative flow cytometry results of CD45+ proportion in the bone marrow (left panel) and peripheral blood of transplanted mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. (D), Real-time PCR analysis of CD34 mRNA expression in the bone marrow (left panel) and peripheral blood of mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. Results shown as mean±SEM (n=3 biological replicates, *P<0.05, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection, Immunofluorescence, Flow Cytometry, Labeling, Transplantation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 6. Detection of BCL11A gene modification and HBG/HBB expression after transplantation of edited human CD34+HSPCs. (A), Representative Sanger sequencing of BCL11A from bone marrow (left panel) and peripheral blood (right panel) of transplanted mice, evidenced from C-T conversions. (B), (C), Real-time PCR was used to detect human HBG and HBB expression in the bone marrow (B) and peripheral blood (C) of SCID mice at 10, 12, 14weeks after transplantation of edited human CD34+HSPCs.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Modification, Expressing, Transplantation Assay, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 7. Comparison of our novel strategy and conventional strategy in maintain CD34 property in vivo. (A), Schematic illustration of protocol. The top panel is the process of conventional strategy IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection, while the below panel is the process of our novel strategy SNA⋅SMNP⋅CBE3 ⋅intraosseous injection. After transplantation edited human CD34+HSPCs with different treatment at 14 weeks, SCID mice from the two different groups were euthanatized, mononuclear cells were resorted and enriched from the bone marrow of mice in both groups. Then, transcriptome analysis was performed to detect and compare the gene expression profiles in each group with naïve CD34 controls. (B), Representative flow cytometry results of CD45+ proportion in the bone marrow of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection counterparts. (C), Real-time PCR analysis of human HBG expression in the bone marrow (left panel) and peripheral blood (right panel) of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅ intravenous (tail vein) injection counterparts. (D), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: IMS⋅EP⋅CBE3⋅intravenous, SNA⋅SMNP⋅CBE3⋅intraosseous injection and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups (right panel). (E), Representative CD34 expression by Real-time PCR in the three groups: SNA⋅SMNP⋅CBE3⋅intraosseous group, IMS⋅EP⋅CBE3⋅tail vein injection counterparts, as well as naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, In Vivo, Injection, Transplantation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Control, Quantitative Proteomics

Journal: Journal of medical genetics

Article Title: Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms.

doi: 10.1136/jmedgenet-2011-100674

Figure Lengend Snippet: Figure 1 (A) Pedigree and mutational analysis of index patient UPN 5869. The pedigree indicates a hereditary breast and ovarian cancer syndrome. Electropherograms illustrate the BRCA1 c.3112G/T mutation (arrow) as well as a nearby single nucleotide polymorphism (SNP) in DNA from fibroblasts and CD34+ leukaemic cells. Both mutation and SNP became homozygous in the leukaemic clone. The SNP array reveals a copy number state 1 and loss of heterozygosity (LOH) at the BRCA1 locus on chromosome 17q of CD34+ leukaemic blasts. (B) Pedigree and mutational analysis of index patient UPN 6371. The pedigree is not specific for a certain syndrome. Electropherograms illustrate the TP53 c.845_848dupGGCG mutation in germline as well as somatic DNA. SNP array shows copy number state 1 and LOH at the TP53 locus on chromosome 17p of CD34+ leukaemic cells. Filled symbols, subjects with malignancies; open symbols, asymptomatic subjects; the arrow indicates the index patient; number within symbol corresponds to the number of asymptomatic individuals. Red regions represent losses and violet regions LOH. BlaCa, bladder cancer; BrCa, breast cancer; Ca, cancer of unknown origin; GaCa, gastric cancer; Leuk, leukaemia of unknown type; Pheo, pheochromocytoma; Plas, plasmacytoma; ProCa, prostate cancer; UtCa, uterine cancer; y, age in years at diagnosis.

Article Snippet: CD34+ cells were separated from diagnostic blood or bone marrow specimens using the magnetic activated cell sorting CD34 MicroBead Kit together with MS Columns and MiniMACS Separator (all Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mutagenesis, Biomarker Discovery

Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Molecular alterations on CD34 + -HSPCs and CECs. Mutated gene frequency and Molecular alterations discovered on CD34+-HSPCs ( A ) and CECs ( B ). On the top the frequency of mutated genes, while on the bottom the table with all the mutated genes in each patient.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Comparative Somatic profiling of CD34 + -HSPCs and CECs in PMF patients. ( A ) Molecular profiles of both CEC and HSPC in patients with PMF. The molecular lesions found in the HSPC are in red, while in Green the ones discovered in the CEC. At the top of the table the clinical characteristics of patients, who successfully recovered CEC. ( B ) Mutated genes shared between HSPCs and CECs.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: Oncoimmunology

Article Title: Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

doi: 10.1080/2162402X.2018.1472195

Figure Lengend Snippet: Antigen expression and susceptibility to redirected lysis of patient blasts and analysis after preparative sorting for CD34- bearing cells.

Article Snippet: CD34-positive cells were enriched by positive selection using the commercial human CD34 + Multisort Kit (Miltenyi Biotec MACS sorting kit, cat. #130–058-701) according to manufacturer’s instructions.

Techniques: Expressing, Lysis