Supplementary data

Supplementary Figure S1. The CCL-βAS3, CCL-βAS3-FB, the three different versions of the Ank-insulated LV, the cHS4-insulated LV and the positive control CCL-MND-GFP LV were packaged in 293T cells. Titers were determined by transducing a permissive cell line (HT29 human colon carcinoma) and measuring vector copies (VC)/cell using quantitative PCR (qPCR) with primers to the HIV packaging signal (psi) of the vector proviruses. Averages of two experiments and SD are shown.

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Supplementary Figure S2. Insulator stability. To test the stability of the Ank and cHS4 insulator, PCR reactions were performed using DNA from MEL clones transduced with the corresponding LV. (a) The two gels on the top row show PCR performed in Ank-FR MEL clones. Left, primers to amplify the Ank insulator placed in forward orientation at the 5’ region of the provirus were used. Lane 1-10 show the 10 clones analyzed, negative controls showing primer specificity were used in lane 11 (MEL clone Ank-R-transduced), lane 12 (MEL clone Ank-RR-transduced) and lane 13 (MEL clone FB-transduced). In lane 14, the positive control, the Ank-FR vector plasmid is shown. Right, primers to amplify the Ank insulator placed in reverse orientation at the 3’ region of the provirus were used. Lane 1-10 shows the 10 clones analyzed, negative controls proving primers specificity was used in lane 11 (MEL clone FB-transduced). In lane 12 the positive control of the Ank-FR vector plasmid is shown. The two gels on the middle row show PCR performed in Ank-RR MEL clones. Left, primers to amplify the Ank insulator placed in reverse orientation at the 5’ region of the provirus were used. Lane 1-10 show the 10 clones analyzed, negative controls proving primers specificity were used in lane 11 (MEL clone Ank-FR-transduced), lane 12 (MEL clone Ank-R-transduced) and lane 13 (MEL clone FB-transduced). In lane 14 the positive control, the Ank-RR vector plasmid is shown. Right, primers to amplify the Ank insulator placed in reverse orientation at the 3’ region of the provirus were used. Lane 1-10 shows the 10 clones analyzed, negative controls proving primers specificity was used in lane 11 (MEL clone FB-transduced). In lane 12 the positive control of the Ank-RR vector plasmid is shown. The gel on the bottom shows PCR performed in Ank-R MEL clones. Primers to amplify the Ank insulator placed in reverse orientation at the 3’ region of the provirus were used. Lane 1-10 show the 10 clones analyzed, negative controls proving primers specificity was used in lane 11 (MEL clone FB-transduced). In lane 12 the positive control, the Ank-FR vector plasmid is shown. The cartoon on the bottom right corner represents the primer designed and expected amplicons sizes. (b) All the clones analyzed on (a) were screened by PCR for the psi region common for all the constructs to confirm the presence of the provirus in all of the clones. For the first two gels, positive controls were lane 11 with an FB-transduced MEL clone and lane 12 with the Ank-RR vector plasmid. In the gel for the Ank-R clones, only the Ank-RR plasmid control is shown. (c) The first gel from the left shows PCR performed to amplify the cHS4 insulator in the 5’LTR. Lane 1-10 show the 10 clones analyzed. The second gel from the left shows PCR performed to amplify the cHS4 insulator in the 3’LTR. Lane 1-10 show the 10 clones analyzed. The last gel, shown on the right, shows that all the clones analyzed were positive for the psi region common for all the constructs. The bands on lane 11, 12 and 13 represent three Ank-LV clones used as positive control. The cartoon on the bottom left corner represents the primer designed and expected amplicons sizes. NTC, no template control.


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Supplementary Figure S3. βAS3-globin expression in erythrocytes differentiated in vitro from human SCD BM CD34+ cells. (a) VCN in transduced SCD BM CD34+ samples cultured in erythroid differentiation conditions. The VCN achieved by transduction with the FB, Ank-R, and cHS4 LV in 4 independent transductions of different SCD donors are shown (SCD-BM donor 1, circle; SCD-BM donor 2, square; SCD-BM donor 3, triangle; SCD-BM donor 4, diamond). Overall differences were assessed by one-way ANOVA with pair-wise comparisons FB vs. cHS4 and Ank-R vs. cHS4, p ≤ 0.02. (b) Percentage of the βAS3-globin mRNA produced and normalized to the total β-like transcripts and to the VCN shown in a. The amount of βAS3-globin mRNA is calculated as the percentage of βAS3-globin transcripts of the total β-globin-like transcripts (sickle β-globin + normal β-globin + βAS3-globin), then normalized to the VCN of the sample (n=4). No significant differences were found among the three groups by one-way ANOVA) (p>0.05). Bars represent average values and SD.

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Supplementary Figure S4. Engraftment in blood and BM from primary mice. Donor cell engraftment was measured by flow cytometry in blood samples from primary mice 10 weeks post-transplant (a) and in BM samples at the day of the harvest 20 weeks post-transplant (b). No differences in engraftment were found between groups either in blood (p=0.69) or BM (p=0.20) (One-way ANOVA). Bars represent average values and SD for each group.

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Supplementary Figure S5. βAS3-globin expression over time in blood from primary recipient mice. (a) Average VCN in the blood of mice transplanted with BM transduced with the FB LV (in black, n=9) and the Ank-R LV (in white, n=8) at the indicated time points post-transplant. No difference in VCN were observed between groups (p=0.16) (Repeated measure ANOVA). (b) Percentage of βAS3-globin mRNA per VCN from the samples shown in a and at the indicated time points. No differences were found between groups (p=0.51) (Repeated measure ANOVA). Average values and SD are shown.

Supplementary Figure S6. BM analysis in primary recipient mice 20 weeks post-transplant. Average VCN is shown on the left y-axis of the graph. The percentage of βAS3-globin mRNA normalized to the β-globin murine mRNA and per VCN, on the right y-axis. FB LV (in black, n=9) and Ank-R LV (in white, n=8). No differences between groups were found for VCN (p=0.91), nor for βAS3-globin RNA per VCN (p=0.11) (t-test). Average values and SD are shown.

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Supplementary Figure S7. Engraftment in blood and BM from secondary mice. Donor cell engraftment was measured in blood samples (a), and in BM samples (b) at the day of the harvest 11 weeks post-transplant. No differences in engraftment were found between groups either in blood (p=0.92) or BM (p=0.56) (one-way ANOVA). Bars represent average values and SD for each group.

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