Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: AsCas12a is superior to SpCas9 with regard to editing HBG1/2 promoters (A) Target sequences for the AsCas12a and SpCas9 RNPs targeting the distal TGACCA BCL11A binding motif at the HBG1/2 promoters. (B) Healthy donor CD34 + cells were electroporated with AsCas12a RNP complexed at 2:1 gRNA to protein ratio or SpCas9 RNP complexed at 4:1 gRNA to protein ratio at the concentration indicated. Indel rates at 1 day after electroporation were determined. N values were n = 8–10 per concentration for AsCas12a, and n = 4–7 per concentration for SpCas9. Four donors were used for AsCas12a and six were used for SpCas9 (see ). (C) Indel profiles of AsCas12a- or SpCas9-edited CD34 + cells. Shown are the normalized profiles (each with a maximum value of 1) for the detected deletions at each base on the target region. n = 4 for AsCas12a and for SpCas9. The red bar marks the distal BCL11A binding motif (TGACCA). (D) Healthy donor CD34 + cells electroporated with 4–8 μM AsCas12a or SpCas9 RNP and placed in erythroid differentiation conditions for 18 days. HbF was measured using reverse-phase ultra-high-performance liquid chromatography, where individual globin chains were eluted. HbF percentage is calculated as γ/(γ+β). N values were n = 22 for control, n = 16 for AsCas12a, and n = 13 for SpCas9. Ten donors were used for the control, 7 for AsCas12a, and 4 for SpCas9 . (E) Healthy donor CD34 + cells electroporated with 4–8 μM AsCas12a or SpCas9 RNP and placed in erythroid differentiation conditions. Indel rates were determined at day 14 erythroid culture. N values were n = 8 for control, n = 12 for AsCas12a, and n = 11 for SpCas9. (F) Number of on- and off-target cut sites identified using Digenome-seq for 25 randomly selected matched sites plus the HBG1/2 target site for AsCas12a and SpCas9. The generalized target sequence and the PAM sequences for AsCas12a and SpCas9 are depicted. Each dot represents one sample. gRNA, guide RNA; HbF, fetal hemoglobin; Indel, insertions or deletions; RNP, ribonucleoprotein. Horizontal bar represents the mean value of the respective treatment group for (B)–(D). Two-way ANOVA was used for (B) and (F), and one-way ANOVA with Tukey’s multiple comparison tests was used for (C) and (D) to determine whether the mean values of the different treatment groups were statistically significantly different. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Binding Assay, Concentration Assay, Electroporation, High Performance Liquid Chromatography, Control, Sequencing, Comparison

Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: AsCas12a edits HSPCs efficiently and leads to edited allele-dependent HbF upregulation (A) Healthy donor CD34 + cells were electroporated with AsCas12a RNP at 3–8 μM. At day 2 after electroporation, common myeloid progenitor (CMP, CD34 + CD38 + CD123 + CD45RA − ), multi-potent progenitor (MPP, CD34 + CD38 low CD90 − CD45RA − ), and hematopoietic stem cells (HSCs, CD34 + CD38 low CD90 + CD45RA − ) were flow-sorted, and indel levels were determined. Indel levels were determined at day 2 after electroporation; n = 3. (B) The proportion of cells with deletion of the 4.9-kb intervening region between HBG1 and HBG2 was evaluated in the total CD34 + cells or subpopulations at day 2 after electroporation using a droplet digital PCR assay; n = 3. (C) Healthy donor CD34 + cells were electroporated without RNP or with 2 μM AsCas12a RNP, and individual cells were cultured in erythroid differentiation conditions. Individual erythroid clones were genotyped and HbF levels were measured by reverse-phase ultra-high-performance liquid chromatography. HbF percentage is calculated as γ/(γ+β). HbF levels are collated based on the number of HBG alleles with indels that each clone had. The dotted horizontal line marks 30% HbF expression. Each dot represents one sample; solid horizontal lines represent the mean value of each group. N values were n = 233 for control and n = 180 for edited. PCR, polymerase chain reaction. Repeat-measures one-way ANOVA with Dunnett’s multiple comparison test was used for (A) and (B), and one-way ANOVA with Dunnett’s multiple comparison test was used for (C) to determine whether the mean values of the different groups were statistically significantly different. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Electroporation, Digital PCR, Cell Culture, Clone Assay, High Performance Liquid Chromatography, Expressing, Control, Polymerase Chain Reaction, Comparison

Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: Efficient editing of SCD CD34 + cells by AsCas12a results in amelioration of disease phenotypes in their erythroid progeny CD34 + cells from four donors with SCD were electroporated with 6 μM of the HBG1/2 -targeted AsCas12a RNP. One SCD sample had a sufficient number of cells for two independent electroporations. (A) Indel levels were measured at day 3 after electroporation; n = 5. (B) CD34 + cells were placed in erythroid differentiation conditions for 18 days, and HbF was measured by reverse-phase ultra-high-performance liquid chromatography. HbF percentage is calculated as γ/(γ+β); n = 5. (C) Cultured SCD erythrocytes were incubated with sodium metabisulfite, and the percentage of cells that sickled was determined; n = 4. (D) Cultured SCD erythrocytes were analyzed on a LoRRca ektacytometer to measure deformability under shear stress, expressed as elongation index, when subjected to decreasing levels of oxygen. The point of sickling, representing the relative oxygen pressure when the SCD RBCs started to sickle during deoxygenation, is plotted for each SCD RBC sample; n = 5. (E) The minimum elongation index of each SCD erythrocyte sample, approximating the flexibility of erythrocytes when deoxygenated, is shown; n = 5. (F) Rheological behavior of cultured RBCs under varying concentrations of oxygen evaluated using a microfluidic platform. The percentage velocity drop was calculated based on the differences between velocity at a specified oxygen concentration and at atmospheric levels of oxygen (21%). N values were n = 6 untreated SCD samples, n = 6 RNP-treated SCD samples, and n = 4 untreated normal samples. RBC, red blood cell; SCD, sickle cell disease. For (A)–(E), each dot represents one sample, and the horizontal line represents the mean value for the treatment group. For (C)–(E), each line connects the untreated and RNP-treated cells from the same donor in the same experiment. For (F), the mean ± standard deviation is shown for each treatment group at the specified oxygen tension. Unpaired Student’s t test was performed for (B), paired Student’s t test was performed for (C)–(E), and two-way ANOVA was performed for (F) to determine whether the differences between edited samples and unedited samples are statistically significant. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Electroporation, High Performance Liquid Chromatography, Cell Culture, Incubation, Shear, Concentration Assay, Standard Deviation

Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: Efficient editing of TDT CD34 + cells by AsCas12a results in improved erythropoiesis and increased hemoglobin content CD34 + cells from three donors with TDT were electroporated with 6 μM of the HBG1/2 -targeted AsCas12a RNP. Their genotypes were donor 1 (compound heterozygous β 0 /β + ), donor 2 (homozygous β 0 /β 0 ), and donor 3 (compound heterozygous β 0 /β + ). See more details in . (A) Indel levels were measured at day 3 after electroporation; n = 4, as each TDT sample had sufficient cells for two independent electroporations. (B) TDT CD34 + cells were cultured in erythroid differentiation conditions, and the expression of erythroid marker CD235a was measured by flow cytometry throughout the culture. (C) Terminal maturation of TDT erythroid cells was determined based on the frequency of erythroid cells that have expelled their nucleus by flow cytometry analysis throughout the culture. (D) Frequency of TDT erythroid cells that were non-viable throughout the course of the 18-day culture was determined by flow cytometry. (E) Globin mRNA levels were quantified and normalized to GAPDH levels by NanoString nCounter in day 14 erythroid cells. (F) Globin chains in day 18 erythroid cells were determined by reverse phase ultra-high-performance liquid chromatography. The amount of globin chain and total hemoglobin content per cell was calculated by plotting the area under the curve against a standard curve generated using a titration series of lysed RBCs with known hemoglobin concentration. TDT, transfusion-dependent β-thalassemia. Each dot (A) represents one independent electroporation. (B)–(D) Mean ± standard deviation of technical triplicates at each time point. For (B)–(D), three independent TDT donors were tested, and data from one representative donor are shown. Data from two additional TDT donors can be found in , and from three additional healthy donors in . Each dot in (E) and (F) represents the value from each technical replicate. The horizontal lines in (A), (E), and (F) represent the mean values of the treatment group. Two-way ANOVA was performed for (B)–(D), and Student’s t test was performed for (E) and (F) on each TDT donor to determine whether the differences between edited samples and unedited samples are statistically significant. ∗∗∗∗ p < 0.0001.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Electroporation, Cell Culture, Expressing, Marker, Flow Cytometry, High Performance Liquid Chromatography, Generated, Titration, Concentration Assay, Standard Deviation

Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: AsCas12a-edited healthy donor CD34 + cells retain HSC functionalities and generate erythroid cells with high levels of HbF expression in vivo Healthy donor CD34 + cells were electroporated with AsCas12a RNP at the concentration indicated, and each NBSGW mouse received 1 × 10 6 cells via intravenous tail vein injection. Animals were euthanized at 16 weeks after engraftment. (A) Indel levels were determined in the CD34 + cells at 1 day after electroporation prior to infusion (pre) and in mouse BM. (B) Indel levels were determined in human CD19 + B cells, CD15 + neutrophils, CD235a + erythroid cells, and Lin − CD34 + HSPCs sorted from mouse BM. (C) Data from three independent in vivo experiments were compiled and correlation analyses were conducted for B cells, erythroid cells, or neutrophils against HSPCs. The black angled line represents the identity line (y = x). (D) The 4.9-kb deletion levels were determined in the CD34 + cells at 1 day after electroporation prior to infusion (pre) and in mouse BM. (E) Human chimerism percentage calculated as hCD45 + /(h+m)CD45 + in BM. (F) Human lineage distribution in BM. B cell percentage is calculated as hCD19 + /hCD45 + , neutrophil percentage is calculated as hCD15 + /hCD45 + , erythroid percentage is calculated as hCD235a + /total cell, and HSPC percentage is calculated as hCD34 + /hCD45 + . (G) Mouse BM was plated in semi-solid methylcellulose media to evaluate the frequency of colony-forming unit granulocyte and monocyte (CFU-GM), burst-forming unit erythroid (BFU-E), and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM). (H) HbF expression by human erythroid cells flow sorted from mouse BM. HbF was measured using reverse-phase ultra-high-performance liquid chromatography, where individual globin chains were eluted. HbF percentage is calculated as γ/(γ+β). (I) Frequencies of HbF + human erythrocytes in mouse BM as determined by immunostaining. For (A)–(I), each bar represents one pre-infusion sample; each dot represents one mouse sample. Horizontal bar represents the mean value of the respective treatment group. For (E)–(I), one-way ANOVA with Dunnett’s multiple comparison test was performed for experiments 1 and 2, and Student’s t test was performed for experiment 3 to determine whether the mean values of the treatment groups were statistically significantly different from the control groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Expressing, In Vivo, Concentration Assay, Injection, Electroporation, High Performance Liquid Chromatography, Immunostaining, Comparison, Control

Journal: Molecular Therapy

Article Title: Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT

doi: 10.1016/j.ymthe.2025.09.031

Figure Lengend Snippet: AsCas12a-edited healthy donor CD34 + cells lead to polyclonal engraftment in irradiated NSG mice Three batches of healthy donor CD34 + cells were electroporated with 8 μM of the HBG1/2 -targeted AsCas12a RNP, intravenously infused into irradiated NSG mice, and followed for 20 weeks. Unique indels were identified and tracked in WB throughout the course of the experiment and in BM at the necropsy. (A) Number of unique indels that were detected at a frequency of ≥0.1% of the reads. (B) Number of unique indels that were detected at a frequency of ≥1% of the reads. (C) Combined frequency of the top two indels. Data are shown as mean ± standard deviation. Groups 1, 3, and 5 received unedited control cells; therefore, data are not shown. BM, bone marrow; F, female (shown as open circle); M, male (shown as closed circle); Pre, pre-infusion sample (shown as closed square); WB, whole blood. Numbers that precede gender denote experiment week, with the week of infusion defined as experiment week 1. n = 12–16 per gender.

Article Snippet: BM cells were collected and stained with anti-mouse CD45-PE-CF594 (BD Biosciences), anti-human CD34-allophycocyanin (APC), anti-human CD19-PE, anti-human CD15-PE-Cy7, anti-human CD45-APC-Cy7 (all from BioLegend), and anti-human CD235a-FITC (Agilent).

Techniques: Irradiation, Standard Deviation, Control