SUPPLEMENTARY MATERIAL

Supplementary Table 1.

Vector / Cumulative MOI / Repeat #1 / Repeat #2 / Repeat #3 / Mean VCN
Mock / - / 0 / 0 / 0 / 0
RSF91 / 80 / 6.12 / 5.41 / 3.91 / 5.15
lv-SF / 340 / 2.74 / 2.83 / 2.91 / 2.83
IL-12 / 60 / 6.78 / 7.88 / 4.58 / 6.41
IL-12 / 60 / 6.01 / 8.37 / 6.52 / 6.97
IL-12 / 60 / 7.22 / 7.24 / 4.12 / 6.19

Supplementary Table 2.

Sample ID / MOI / % LNGFR+ cells (transduction efficiency) / VCN/genome / Secreted hIL-12 level
(pg/2hr/106 cells)
130278 / 0 / 0.37 / 0 / 9.8
130278 / 10 / 18.0% / 1.4 / 24.0
150549 / 0 / 0.72 / 0 / 15.1
150549 / 10 / 33.6% / 1.2 / 92.3

Supplementary Figure Legends

Supplementary Figure S1. PBMCs isolated from AML patient blood samples contain a major population of AML blasts. PBMCs were isolated from AML patients’ fresh blood samples and stained with 7-AAD and antibodies against human CD45 and CD14. Stained samples were subjected to flow cytometry analysis and live cells (7-AAD-negative) were first selected, then gated by CD45 and Side Scatter (SSC). Lymphocytes (Lym) were separated as a low-SSC, high-CD45+ population. The population consisting of AML blasts (Blasts) and monotytes (Mono) was further distinguished by CD14-staining. Gran: granulocytes.

Supplementary Figure S2. Ex vivo culture and LV-transduction of patient primary PBMCs were targeted on AML blasts. (a) PBMCs were isolated from AML patients’ blood samples and an aliquot was stained with 7-AAD, and antibodies against human CD45 and CD14 to examine cell phenotype. The remaining cells were cryopreserved, thawed and cultured in complete growth medium as described in Materials and Methods. After 5 days of culture, cells were harvested and stained with antibodies against human CD45 and CD14 and 7-AAD. Lym, lymphocytes; Mono: monocytes. (b) PBMCs from AML patient (#130012) blood sample was isolated, cryopreserved, thawed (on day 1) and transduced (on day 3) with LV/IL-12 at MOI of 10. Cells were collected at three days after transduction (on day 6) and stained with 7-AAD, and antibodies against human CD45 and LNGFR (CD271) to examine cell phenotype.

Supplementary Figure S3. Effect of different sera on primary AML cell survival. Primary AML cells from 7 patients were thawed and cultured in complete AIM-V® medium, supplemented with either human AB serum, bovine serum, or horse serum for up to 8 days. Viable cell numbers were quantified at the time of thawing and every two days later. Each patient cell survival curve was plotted in an individual graph (with patient unique identifier number indicated on top of each graph).

Supplementary Figure S4. Comparison of effects of human serum and KnockOutTM Serum (KOSR) on primary AML cell growth. Primary AML cells from 4 patients were thawed and cultured in complete AIM-V® medium supplemented with either human serum or KOSR for up to 8 days. Cells were cultured in either flat (a) or round (b) bottom culture plates. Viable cell numbers were quantified at the time of thawing and days 2, 4, 8 after thaw. Each patient cell survival curve was plotted in an individual graph (with patient unique identifier number indicated on the top of each graph).

Supplementary Figure S5. Vector-transduced cell growth in the IVIM study. Mouse lineage marker-negative BM cells (Lin-) were thawed and seeded in 24-well plates at 1x105 cells per well. Cells were transduced with different viral vectors and cultured for up to 15 days (as described in the Materials and Methods section). Samples at each indicated time point were counted either with or without a 1:10 dilution for Trypan blue exclusion. The total live cell number was plotted. Data shown are mean ± SD from 3 to 9 independent experiments.

Supplementary Figure S6. Stability of LV-transduced primary patient AML cells in various buffer conditions. AML samples isolated from two patients were cultured and transduced with LV/IL-12 (as described in Materials and Methods). The final transduced cell products were washed and resuspended in Plasma-Lyte A in the absence (0%) or presence of increasing percentage of human serum. Cell numbers were counted by Trypan blue exclusion before (Input) and after wash and resuspension (Wash/Resuspension) in the indicated buffer. The cell product was stored in sterile conditions at room temperature overnight and cell number was counted again.

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