CD34+ cells transduced with MGMT* lentiviral vectors show transient chemoprotection after in vivo treatment with BG and TMZ.
We investigated whether in vivo chemoprotection could be achieved after transduction with MGMT*-expressing lentiviral vectors based on SIV and autologous transplantation of mobilized peripheral blood (PB) or BM CD34+ cells in 5 rhesus macaques (Table ). Gene transfer efficiency in the CD34+ cell population before transplant in rhesus macaques varied between 18% and 53%, as determined by flow cytometry (Table ).
Reconstitution of irradiated rhesus macaques with transduced, autologous CD34+ cells
In a first set of experiments, AMD3100-mobilized PB CD34+ cells from animals RQ4876 and RQ4795 were transduced using SIV vectors expressing GFP-MGMT* fusion protein (Figure A). We administered 2 daily doses of 500 cGy total body irradiation before infusion of 5.6–6.4 × 106 CD34+ cells/kg. Hematopoietic recovery was documented between 7 and 17 days (Table ). At 3–6 months after transplant, each animal received the first of 4 successive BG/TMZ treatments given at 1- to 2-month intervals. Substantial myelosuppression was seen in both animals after drug administration (Figure A).
In vivo selection in SIV-transduced animals RQ4876 and RQ4795 after multiple cycles of BG/BCNU and/or BG/TMZ.
Animal RQ4876 showed a rapid increase in transduced PB cells of all lineages in vivo after drug treatment. Steady-state marking levels of 2%, 6%, and 5% in granulocytes, lymphocytes, and monocytes, respectively, increased to peak levels of 52%, 33%, and 36% in the respective lineages after the first (granulocytes), second (lymphocytes), or third (monocytes) dose of BG/TMZ. The fourth dose of BG/TMZ led to only moderate increases in GFP+ cells in all lineages. Treatment with BG/TMZ was well tolerated, with little to no cumulative toxicity. The proportion of GFP+ cells subsequently declined gradually over a period of 200 days after the last BG/TMZ administration; marking levels in PB stabilized to values similar to those observed at steady state before the first treatment with BG/TMZ, with 2% GFP+ granulocytes, 6% GFP+ lymphocytes, and 3% GFP+ monocytes (Figure B). Lentiviral vector provirus copy numbers were also determined by real-time PCR at several time points in DNA from PB (data not shown). Overall, there was a good correlation between flow cytometry and real-time PCR results, which suggests that on average, repopulating cells contained no more than 1 vector copy and that gene silencing was not responsible for the gradual decrease in gene marking levels in vivo.
Animal RQ4795 had very low steady-state gene marking levels in PB granulocytes (0.1%), lymphocytes (0.01%), and monocytes (0.2%; Figure B). We tested the safety and feasibility of improving very low baseline gene marking levels with in vivo infusion of BG/TMZ. No change in GFP+ granulocytes was detected at any time point after BG/TMZ administration. A peak increase in GFP+ lymphocytes (0.2%) and monocytes (1.5%) was observed after the second dose of BG/TMZ, but the increase was only transient, returning to baseline levels within 30 days. The third and fourth doses of BG/TMZ resulted in substantial myelosuppression, but had no impact on gene marking levels in any lineage.
In a second set of experiments, we tested whether alternative SIV MGMT*-expressing vectors in combination with minor modifications to transduction conditions and escalating doses of TMZ could result in improved longer-term gene marking levels. Rhesus competitive repopulation experiments were set up using equal numbers of mobilized PB or BM CD34+
cells transduced with SIV vectors expressing MGMT* alone (containing GFP or yellow fluorescent protein [YFP]) or MGMT*-HOXB4-GFP (see Methods and Figure ). Multicistronic cassettes containing GFP or YFP were generated in each vector using the 2A peptide from porcine teschovirus–1 (P2A) and thosea asigna virus (T2A) (Figure , B–D). All 3 animals in this study — RQ4513, RQ4099, and RQ4152 — were mobilized with G-CSF plus SCF and received 2- to 3-fold more CD34+
cells/kg compared with the AMD3100-mobilized animals RQ4876 and RQ4795 after total body irradiation conditioning (Table ). Hematopoietic reconstitution was rapid, with the absolute neutrophil count (ANC) reaching 500 cells/μl or more by 8–11 days after transplant. All animals showed a total of 3%–6% marked PB granulocytes and monocytes, and 1%–12% marked PB lymphocytes, at 60 days after transplant (Figures , , ). After stable engraftment, we evaluated the drug combination BG/TMZ. The dose of BG was fixed at 120 mg/m2
, but, in contrast to animals RQ4876 and RQ4795, the dose of TMZ was gradually escalated from 170 to 450 mg/m2
/d for a 5-day period. All animals showed significant myelosuppression after each treatment (Supplemental Figure 1; supplemental material available online with this article; doi:
In vivo selection in SIV-transduced animal RQ4513 after 2 cycles of BG/TMZ and 2 cycles of BG/BCNU.
In vivo selection in SIV-transduced animal RQ4099 after 1 cycle of BG/TMZ.
In vivo selection in SIV-transduced animal RQ4152 after multiple cycles of BG/TMZ and 1 cycle of BG/BCNU.
The first animal in this group, RQ4513, was treated with 2 courses of BG/TMZ at 12 and 15 weeks after transplant. The ANC nadirs were 780 cells/μl at day 16 after the first treatment and 210 cells/μl at day 14 after the second treatment. The percentage of GFP+ and YFP+ granulocytes (Figure A) and monocytes (Figure C) increased from 4% to 40% (YFP) and 4% to 50% (GFP), but gradually declined back to 4% (GFP) and 10% (YFP) over a period of 50 days after the second dose of BG/TMZ. The absolute number of YFP-transduced granulocytes increased from 40 cells/μl before drug treatment to approximately 200 cells/μl after 2 courses of BG/TMZ, which suggests that YFP-transduced CD34+ cells were protected and expanded after BG/TMZ treatment (Figure B). Similarly, the percentage of GFP+ and YFP+ lymphocytes doubled after the second BG/TMZ administration, but GFP+ lymphocytes rapidly declined back to pretreatment levels (Figure D).
The second animal, RQ4099, was treated with a single course of BG/TMZ at week 10 after transplant. The ANC nadir was 190 cells/μl at day 14 after treatment. The percentage of GFP+ and YFP+ granulocytes and monocytes increased from a steady-state level of 3%–5% to a peak of 25%–30%, but returned to baseline gene marking levels within 50 days after BG/TMZ administration (Figure A). In vivo enrichment of GFP+ and YFP+ B cells was also transient (Figure B). In T cells, GFP and YFP marking levels were 1% at steady state and peaked at 13% (GFP) and 23% (YFP) after BG/TMZ treatment. The proportion of gene marked T cells subsequently declined and stabilized at 4% (GFP) and 5% (YFP) (Figure B). The animal died approximately 3 months after BG/TMZ treatment due to pulmonary fibrosis. This complication is occasionally seen in animals not treated with BG/TMZ and is likely secondary to radiation received as a conditioning regimen prior to transplant.
The third animal, RQ4152, had pretreatment gene marking levels of 5% in granulocytes, 3% in monocytes, 10% in B cells, and 1% in T cells. The animal was treated with 7 courses of BG/TMZ, with TMZ doses ranging from 170 to 450 mg/m2
/d. The ANC nadir was 850 cells/μl at day 6 after the first treatment (170 mg/m2
TMZ) and 7 cells/μl at day 9 after the last treatment (450 mg/m2
TMZ). After each course of drug treatment, there was a rapid increase in GFP marking in all lineages. The highest marking achieved after the last course of BG/TMZ treatment was 94% for myeloid cells and 57% for lymphoid lineages (Figure A). The proportion of GFP+
cells subsequently declined and leveled off at 10% for granulocytes, 5% for monocytes, 17% for B cells, and 30% for T cells at 150 days after the last treatment (Figure A). Correspondingly, the absolute number of transduced granulocytes increased after each drug treatment with, for example, levels of 80 cells/μl before the fourth course peaking to 698 cells/μl after the seventh course and stabilizing at approximately 250 cells/μl (Figure B). These results indicate that MGMT*-transduced cells were protected and expanded following multiple courses of BG/TMZ treatments with TMZ dose escalation up to 450 mg/m2
. In pediatric patients, the maximum tolerated dose of TMZ without BG is 185 mg/m2
); therefore, gene therapy resulted in substantial dose escalation due to protection from hematopoietic toxicity. As shown by pharmacokinetic data (Table ), the absorption and clearance of TMZ in animal RQ4152 at the time of the seventh dose of TMZ was comparable to that seen in treated pediatric patients. Moreover, the AUC for TMZ was about 2-fold greater than that seen in pediatric patients (Table ), confirming that BM protection was achieved against BG/TMZ by MGMT* overexpression in this animal. This is particularly interesting given that BG is known to significantly enhance TMZ toxicity, necessitating TMZ dose reductions (30
). Given the relative lack of nonhematopoietic toxicity seen in our series with BG/TMZ, we conclude that hematopoietic protection via transfer of MGMT* allows for substantial dose escalation and administration of repeated drug courses.
Pharmacokinetic study of RQ4152 after the seventh course of TMZ/BG treatment
Polyclonal hematopoiesis is sustained after BG/TMZ treatment.
The clonal composition before and after BG/TMZ treatment was analyzed by linear amplification–mediated PCR (LAM-PCR) in animals RQ4152 and RQ4513, which showed the highest in vivo gene marking levels. DNA isolated from PBLs before and after 1, 2, 6, and 7 courses of BG/TMZ was used for LAM-PCR. Gel electrophoresis showed contribution of multiple clones at all time points analyzed (Supplemental Figure 2). Individual bands from LAM-PCR products were picked from the gel and sequenced directly after PCR amplification. Using previously described selection criteria (31
), we retrieved and analyzed 254 independent unequivocal retroviral integration sites (ISs; 152 for RQ4152, Supplemental Table 1; and 102 for RQ4513, Supplemental Table 2). The majority of these ISs occurred within transcription units, mainly within introns (Table ), consistent with data from previous reports (31
). Also, in agreement with previously published data (31
), SIV integration events did not favor locations upstream or downstream of transcription units (Table ), indicating no preference for integration near transcription start sites. In animals RQ4152 and RQ4513, 7.9% and 3.9% of ISs identified, respectively, were in or near cancer-associated genes (CAGs; Table and Supplemental Tables 1 and 2). We identified 8 clones that appeared repetitively before and after drug treatment, which suggests that the same clones contributed to hematopoiesis after in vivo selection with BG/TMZ.
IS distribution and association with CAGs for RQ4152 and RQ4513
In vivo treatment with BG/BCNU does not result in long-term chemoprotection and causes high levels of nonhematopoietic toxicity.
In animals RQ4876, RQ4513, and RQ4152, which showed evidence of increased gene marking after multiple BG/TMZ treatments, we tested whether chemoprotection could be achieved using BCNU as an alternative alkylating agent. Several months after the last dose of BG/TMZ, we administered 1–3 successive infusions of 120 mg/m2 BG and 20 mg/m2 BCNU (hereafter referred to as BG/BCNU). In animal RQ4876, a moderate but steady increase in marking levels was seen after each BG/BCNU dose. Pretreatment levels of 2% in granulocytes, 6% in lymphocytes, and 5% in monocytes peaked at 9%, 17%, and 10%, respectively, after the third dose (Figure B). BG/BCNU treatment also resulted in a rapid increase in GFP/YFP marking in all hematopoietic lineages in animal RQ4513, with maximal levels of 90% in granulocytes and monocytes, 60% in B cells, and 40% in T cells (Figure ). A more modest effect was seen in animal RQ4152 (Figure ).
All animals that received BG/BCNU eventually developed substantial nonhematopoietic toxicity, in contrast to what was seen using BG/TMZ. RQ4876 developed severe tachypnea, fevers, and failure to thrive with weight loss 1 month after the third dose of BG/BCNU. X-rays revealed diffuse pulmonary infiltrates consistent with pneumonitis (Supplemental Figure 3A). Euthanasia was performed 35 days after the last administration of BG/BCNU. Animal RQ4513 developed severe pulmonary and gastrointestinal complications, and euthanasia was performed 2 weeks after the second cycle of BG/BCNU. Specimens collected at necropsy revealed extensive pulmonary edema and widespread colonic necrosis with pseudomembrane formation (Supplemental Figure 3B). Animal RQ4152 was clinically well after receiving 7 courses of BG/TMZ, but developed severe gastrointestinal complications and died 7 days after 1 course of BG/BCNU. At necropsy, specimens were consistent with a diagnosis of severe necrohemorrhagic colitis (Supplemental Figure 3C). While lower doses of BCNU may have been less toxic, we observed no significant decrease in complete blood count and no change in vector marking levels when a single test dose of 5 mg/m2 was administered in animal RQ4876 (data not shown).
Because all animals were euthanized shortly after BG/BCNU administration, long-term persistence of improved marking levels could not be assessed. In the BCNU-treated animal RQ4876, follow-up was possible for a slightly longer period of time after BCNU administration (about 80 days); in this animal, a clear trend down in marking levels was evident at the time of euthanasia (Figure B), which suggests a lack of persistent chemoprotection with BCNU, as was seen after TMZ administration.
Absence of long-term chemoprotection in vivo is not caused by lack of transduction of long-term repopulating HSCs.
We investigated whether the lack of stable selection in vivo could be the result of poor transduction of long-term repopulating HSCs. Several lines of evidence indicated transduction of HSCs. First, in all animals, persistence of substantial expression of the GFP transgene in T cells, B cells, granulocytes, and monocytes for several months after transplant was an indication of HSC transduction (Figure B and Figures –). Second, in animal RQ4876, 10% of CD34+ cells isolated from the BM 630 days after transplant were GFP+, as assessed by flow cytometry (data not shown). Third, we also obtained molecular evidence of transduction of HSCs in animal RQ4876. Using DNA isolated from the BM of this animal more than 1 year after transplant, we performed LAM-PCR and isolated 10 ISs. Demonstration of common lentiviral ISs in sorted subpopulations of the PB (T cells, B cells, and granulocytes) collected before BG/TMZ treatment (day 60 after transplant), after the last dose of BG/TMZ (day 374), and after the last dose of BG/BCNU (day 630) was performed by PCR using 1 vector long-terminal repeat (LTR) primer and 1 genomic primer spanning a region of each IS. We were able to detect 2 common ISs (Figure ), providing a clear demonstration of the transduction of HSCs contributing in the long term to the different hematopoietic lineages. Interestingly, of the 2 common ISs, the one designated IS 1 (Figure ) was undetectable prior to drug treatment, but its contribution to all lineages increased over time after BG/TMZ and BG/BCNU treatment, a characteristic of HSC dynamics. Overall, these observations rule out the lack of transduction of HSCs as a possible mechanism for the absence of long-term chemoprotection. Given the consistent data among the 5 animals described in the present study, we believe that these conclusions can be generalized to all animals.
Transduction of long-term repopulating rhesus macaque HSCs with lentiviral vectors.
HOXB4 does not confer HSC amplification in the rhesus macaque transplant model.
HOXB4 is a potent HSC amplifier in murine transplantation models. In our competitive rhesus transplantation model, we investigated whether HOXB4 could improve engraftment of transduced HSC and progenitor cells in animals RQ4513, RQ4099, and RQ4152 (Table ). Gene transfer efficiencies in CD34+ cells were similar between MGMT* and MGMT*-HOXB4-GFP vectors before cell infusion based on flow and CFU analysis. For animals RQ4513 and RQ4099, the GFP/YFP ratio allowed direct measurement of competitive engraftment of MGMT*-HOXB4-GFP– versus MGMT*-transduced cells. In both animals, we observed a similar engraftment pattern. The initial engraftment levels for both MGMT*- and MGMT*-HOXB4-GFP–transduced granulocytes at 2 weeks after transplant were similar in RQ4513 (17% versus 13%; Supplemental Figure 4A) and RQ4099 (23% versus 25%; Supplemental Figure 4B). Marking levels gradually declined for both compartments before they stabilized at a similar level. In RQ4513, MGMT*-HOXB4-GFP marking stabilized at 2.6%, and MGMT* marking stabilized at 2.9%, in granulocytes 78 days after transplant (Supplemental Figure 4A). Marking levels in granulocytes in RQ4099 stabilized at 2.5% for MGMT*-HOXB4-GFP and 3.3% for MGMT* at 66 days after transplant (Supplemental Figure 4B). For animal RQ4152, because both MGMT* and MGMT*-HOXB4-GFP–transduced cells used GFP as an expression marker, Southern blot was used to distinguish the 2 compartments. Again, there was no difference in engraftment levels between MGMT*- and MGMT*-HOXB4-GFP–transduced cells (Supplemental Figure 4C, lane 1). Moreover, we did not observe any advantage for in vivo selection in the cells containing the MGMT*-HOXB4-GFP vector. For instance, in RQ4513 (Figure ) and RQ4099 (Figure ), marking levels in all lineages increased in parallel between MGMT*- and MGMT*-HOXB4-GFP–transduced cells after drug treatment. For RQ4152, there was also no difference in marking levels between MGMT*- and MGMT*-HOXB4-GFP–transduced cells after BG/TMZ treatment, as determined by Southern blot (Supplemental Figure 4C, lanes 2–5). At 8 months after transplant, HOXB4 expression was detected in MGMT*-HOXB4-GFP–marked PB cells sorted by flow cytometry (Supplemental Figure 5), which confirmed that the lack of amplification was not caused by absent expression of HOXB4.