Transduction of normal G-CSF mobilized peripheral blood mononuclear cells, lineage depleted mononuclear cells, and CD34+ cells
We transduced cells derived from normal mobilized peripheral blood progenitor cells using a lentiviral GFP vector with an MOI of 10. Using a custom mixture of antibodies to CD3, CD14, CD19 and CD56, we removed cells that expressed these antigens, and our average yield was 16% of the initial starting mononuclear cell population after density depletion. Our average yield of CD34+ cells was 1.6% of the starting mononuclear cell population, and the average purity was 84.4% by flow cytometry. The transduction efficiency by flow cytometry for GFP positive cells was 8.7% for mononuclear cells, 6.4% for lineage depleted cells, and 5.5% for CD34+ cells. However, the transduction efficiency in colony forming cells as assessed by GFP expression was 12.5% for mononuclear cells,16% for lineage depleted cells, and 27.7% for CD34+ cells. The transduction efficiency in colony forming cells as assessed by polymerase chain reaction (PCR) was 12.5% for mononuclear cells, 18.8% for lineage depleted cells, and 10.4% for CD34+ cells. These values for transduction efficiency all represent mean values for the different samples of normal, G-CSF mobilized peripheral blood progenitor cells.
Correction of defective FANCA lymphoblasts using FancA-sW
Human lymphoblasts derived from a FANCA patient were transduced overnight with a FANCA or a GFP-expressing lentiviral vector at a multiplicity of infection (MOI) of 10. The transduced cells were subsequently exposed to increasing doses of MMC and viable cell numbers were obtained 2 and 4 days after initial exposure to MMC (). At Day 2, cell number differences between the lymphoblasts transduced with FancA-sW versus those transduced with the GFP vector were statistically different in all concentrations of MMC. The p values were 0.0015, 0.0034, 0.0091, 0.022 for 5, 10, 20, and 50 nM of MMC, respectively. Likewise, Day 4 also had a statistically different number of viable cells in all MMC concentrations, with higher statistical differences than that observed at Day 2. The p values for Day 4 were 0.0017, 7.8 × 10−5, 1.90 × −5, and .0019 for 5, 10, 20, and 50 nM of MMC, respectively. Without MMC, there was no difference in cell viability between lymphoblasts transduced with the two vectors (p value of 1.00 for both Day 2 and Day 4), suggesting that there was no innate in vitro increase of cell growth or viability in cells transduced with the FancA-sW vector. These studies demonstrated that FancA-sW transduction can correct the hypersensitivity of human FANCA-deficient cells to MMC. These results further suggest that the housekeeping constitutive internal promoter, the PGK promoter, is sufficient to achieve phenotypic correction.
Transduction of defective FANCA lymphoblasts by FancA-sW restores resistance to DNA cross-linking
Hypoxic environment and NAC improve transduction efficiency and viability of human FANCA CFU
One of the major technical difficulties for development of gene therapy for FA is the extraordinary ex vivo fragility of FA bone marrow cells, which is believed to be in part due to oxidative damage. We therefore sought to compare the effect of oxidative stress and length of transduction on the transduction rate and colony forming potential of primary human FA bone marrow. In order to minimize ex vivo manipulation and multiple integration events, bone marrow mononuclear cells were transduced at an MOI of 10. In an initial experiment, the effects of low oxygen, short overnight vector exposure (without pre-stimulation) and the use of NAC were compared to the standard pre-stimulation of 24 hours, followed by 2 daily cycles of transduction in normal oxygen without NAC (data not shown). Bone marrow cells transduced overnight in the presence of both low oxygen and NAC exhibited increased colony number/plate compared to conditions of 21% oxygen without NAC, mean 6.5 vs. 2 colonies per 3 × 104 cells plated (p = 0.12). Short transduction period, use of 5% oxygen and use of NAC were each associated with increased colony number, with p values of 0.15, 0.15, and 0.089, respectively. Using either short transduction or NAC was associated with a trend in having increased GFP-positive colonies (p = 0.10 for both). The use of 5% oxygen showed a significant difference compared to using 21% oxygen, with an average of 2.125 GFP colonies compared to 0.75 colonies (p = 0.018). Lastly, there was a significant difference in the number of GFP vector transduced colonies when the brief transduction, low oxygen with reducing agent was compared to long transduction, 21% oxygen and no NAC, with an average of 4 colonies/plate in the overnight low oxygen with NAC versus 0 colonies/plate in the standard conditions (p = 0.00042).These data demonstrate that short transduction with reduced oxidative stress is more effective in transducing progenitor cells and at least as effective in preserving colony forming cells compared to standard transduction conditions.
In a second experiment, we focused on the role of oxygen and NAC on transduction and colony formation in a single overnight transduction (). The combined use of 1 mM NAC and 5% oxygen showed a significant increase in the number of colonies formed compared to the other conditions (p values all < 0.03). The greatest difference was between the colonies formed in 5% oxygen with NAC and those formed under 21% oxygen without NAC, with an average of 11.5 ± 0.71 (SD) colonies and 3 ± 0.05 (SD) colonies respectively (p = 0.0034). No other group was significantly different from another. Transduction rate, measured by GFP expression was statistically similar in the four groups (p > 0.22); with 60% of colonies placed in hypoxic conditions with NAC being GFP-positive. Combining data from the various groups, the use of NAC was associated with both increased colony formation, 8.25 versus 3 (p value of 0.038), and increase in the number of transduced colonies, 4.25 versus 0.75 (p value of 0.022). Thus the overnight (14-hour) transduction of whole bone marrow, in an environment of reduced oxygen stress, results in a high level of transduction with preservation of progenitor cells.
Hypoxic environment and/or reducing agent improve the transduction efficiency and viability of human Fanconi bone marrow progenitors
Correction of human FA-A bone marrow cell MMC hypersensitivity after FancA-sW transduction
Three human FANCA bone marrow samples were used to analyze efficacy of the FancA-sW lentiviral vector. The mononuclear cells derived from these samples were transduced in 5% O2 with 1mM NAC. The cells were subsequently divided and either plated in methylcellulose or placed into suspension culture in the hypoxia chamber, with or without MMC.
While there is generally a reduced clonogenic capacity of FA hematopoietic progenitors in vitro,18–20
CFU were readily detectable in bone marrow mononuclear cells from three different FANCA
patients transduced with FancA-sW as compared to GFP (mean 10.7, 54 and 14 colonies/3 × 104
cells plated versus 6, 27, and 5 colonies/3 × 104
cells plated, respectively). The distribution of colony size is shown in . Colony morphology was heterogeneous, including colonies greater than 2 mm and clusters of < 50 cells. On average, GFP vector transduced cells produced 52% less colonies and clusters compared to FancA-sW transduced cells (p = 0.019). While number of colonies diminished with increasing MMC concentration, there were statistically more colonies for the FancA-sW transduced cells than the GFP vector transduced cells (). The fraction of CD34+
cells in the patient bone marrow ranged from 0.1 to 0.7%, and did not correlate with colony formation or transduction efficiency. Transduction efficiency ranged from 15 to 90% for both FancA-SW and GFP vector transduced bone marrow by colony PCR, demonstrating effective transduction using a single, overnight transduction at an MOI of 10.
There are more colonies surviving in MMC for FancA-sW transduced human FANCA bone marrow cells than for the control GFP vector transduced cells
Transduced bone marrow cells placed in cell culture were exposed to increasing concentrations of MMC in triplicate wells. Viable cells were subsequently counted at Day 2 and Day 4 with the results shown in . All three samples demonstrated statistically significant increased cell number in the FancA-sW transduced cells, compared to GFP vector transduced controls, at the highest level tested on both Day 2 and Day 4 (). Patient 1 showed significant differences at both 5 and 10 nM of MMC with a 2.1-fold difference in cell number in cells exposed to 10 nM MMC. At Day 4, patient 1 showed significant difference in cell number only in cells exposed to 10 nM MMC. For patient 2, Day 2 samples showed significant differences in cell numbers in all three MMC concentrations tested. On Day 4, patient 2 had significant differences in transduced cells exposed to MMC at both 10 and 20 nM concentrations. Patient 3 had GFP vector transduced cells that were the most resistant of the three samples to the effects of MMC. Overall, these results indicate that human FA bone marrow cell hypersensitivity to MMC-induced DNA cross linking is corrected by transduction of the cells with the FancA-SW vector in hypoxia with reducing agent, although the effect varied between individual patients.
There is superior survival of FancA-sW transduced FANCA bone marrow cells in culture in MMC than control GFP vector transduced cells
p values for differences in numbers of surviving transduced cells for FANCA-sW versus GFP vector with increasing MMC concentration
Isolation and transduction of Fanconi bone marrow derived CD34+ cells
From 1.2 ml of patient bone marrow, we obtained 18 million mononuclear cells after density depletion. From a fraction of 11 million bone marrow mononuclear cells, we obtained 110,000 CD34+ cells after positive selection on the Miltenyi MACS, a yield of 1% of the mononuclear cells. Fifty thousand cells were transduced with the GFP lentiviral vector, and the same number with FancA-sW. After overnight transduction in the hypoxia chamber with NAC, 31,000 (62%) were recovered from the GFP vector transduction, and 35,000 (70%) from the FancA-sW transduction. To extrapolate the yield for a scaled up clinical procedure, for a 500 ml bone marrow harvest, this would correspond to 52,500,420 transduced CD34+ cells, or 1 million transduced bone marrow CD34+ cells/kg for a 50 kg patient, 1.3 million/kg for a 40 kg patient, or 1.7 million/kg for a 30 kg patient. The transduced CD34+ cells exhibited colony growth in methylcellulose, with survival in MMC that was superior to that of transduced mononuclear cells (). For 10 nM MMC, there were 60.7% ± 10.1% of the colonies at 0 nM MMC for the CD34+ cells, and 8.9 ± 7.4% for mononuclear cells (p=0.003), and at 20 nM MMC, there were 35.7% ± 10.3% of the colonies at 0 nM MMC for the CD34+ cells, and 8.9% ± 1.3% for the mononuclear cells (p=0.019), suggesting that there was even better functional correction of the colony forming cells from transduced purified FA patient derived CD34+ cells than the transduced mononuclear cells. By colony PCR, 12.5% of the colonies plated from the FancA-sW transduced CD34+ cells were positive for the transgene without MMC, and 18.8% positive with 10 nM MMC.
Transduced CD34+ FA patient bone marrow cells form colonies and survive mitomycin C (MMC)
Analysis of integration sites by linear amplification-mediated polymerase chain reaction (LAM-PCR)
LAM-PCR was used to analyze insertion sites for FancA-sW transduced normal mobilized progenitor cells and FANCA bone marrow cells. Insertion sites for the normal mobilized progenitor cells are exhibited in . LAM-PCR analysis of DNA from colonies derived from FancA-sW transduced human FANCA bone marrow cells demonstrated 1–2 integrations per CFU, with the majority exhibiting one integration per cell (data not shown). By BLAST analysis, the integration sites were not within coding sequences of the genome.
LAM-PCR Insertion site analysis: 26 insertions in bulk culture of transduced normal CD34+ cells