This study was designed to evaluate the outcome of HSC gene therapy in X-CGD mice conditioned with either 300 cGy or ablative irradiation prior to transplantation with SF71gp91phox
-transduced marrow. Both regimens were effective in achieving long term expression of gp91phox
, confirming previous studies on the efficacy of reduced intensity conditioning regimens for gene therapy of murine X-CGD. 22–25
We also observed the occurrence of higher than expected frequencies of oxidase-positive neutrophils in 300 cGy-conditioned recipients, which we had not seen in submyeloablative conditioning studies using an MSCV-based vector.22–25
Thus, we also compared vector marking and integration sites between the two conditioning regimens, finding differences that suggest that the intensity of irradiation conditioning can influence selection for vector-containing donor cells in post-transplant engraftment and hematopoiesis. This novel finding may have important implications for approaches used to achieve engraftment of genetically corrected human HSC in clinical trials.
For integration site analysis, we chose LM-PCR rather than the more sensitive linear amplification-mediated PCR (LAM-PCR) in order to recover sites that were relatively abundant in the population. There are several points regarding the approach taken in our and similar studies. First, not all RIS are equally recoverable, due to intrinsic limitations in the LM-PCR assay41
and to sequence-dependent differences in cloning efficiency and sequence amplification. Second, hematopoiesis in the murine transplant model is oligoclonal43
and active clones vary over time. For example, serial studies of secondary CFU-S12 find 3 – 9 independent clones present at any one time, with new clones emerging every few months while others disappear.44,45
Third, previous studies in the mouse have largely focused on analysis of spleen,11,12,40
which is predominantly lymphoid cells. While we evaluated spleen, we additionally analyzed non-adherent BM, which are primarily neutrophils and myeloid progenitor cells. Furthermore, our analysis of secondary CFU-S12 allowed for characterization of integrations in clonal populations of primitive multipotential myeloid cells.
We saw both similarities and important differences in SF71gp91phox RIS recovered from primary recipients conditioned with either 300 cGy or 950 –1110 cGy. Similarities include 1) the relative distribution of unique sites identified in spleen, marrow, or both; 2) the percentage of insertions near the TSS and in or near RefSeq genes; 3) vector copy number in secondary CFU-S12; 4) an increased frequency of integrations in or near cancer-associated genes relative to their occurrence in the genome; and 5) a similar enrichment for genes present in an HSC transcriptome database and an over-representation of certain gene ontology categories, most notably terms associated with transcription. We observed three notable differences between the two conditioning cohorts. First, almost twice as many donor CFU-S12 were vector-positive in 300 cGy-conditioned mice. Second, there was a significantly higher frequency of RIS associated with cancer-associated genes in marrow and in multipotential myeloid cells (CFU-S12) from mice conditioned with 300 cGy compared to lethally irradiated mice. Third, the majority of insertions seen in more than one primary recipient, most notably Evi1, were found in the 300 cGy -conditioned cohort. Although we did not track peripheral blood insertion sites in this study, we speculate that the increased fraction of oxidase-positive neutrophils in many 300 cGy-conditioned recipients relative to the 950–1100 cGy cohort also reflects these differences.
In aggregate, our results confirm previous studies in mice, non-human primates, and clinical trials, showing that gamma-retroviral integration sites within engrafted HSCs represent a biased population.4,9,11–15,18
Additionally, our results suggest for the first time that the transplant conditioning regimen may further bias this trend. In partially ablated recipients, where the marrow is a more "competitive" environment for repopulation,25,46
insertions with potential activating effects on proliferation and/or survival may confer an advantage for successful HSC engraftment. Although vector insertion near a CAG may also promote clonal expansion of progenitor cells, it is noteworthy that the relatively greater enrichment in 300 cGy-conditioned mice compared to 950–1100 cGy is present in samples obtained at more than 6 months post-transplant, suggesting that these insertions conferred an advantage at the time of HSC engraftment.
It seems unlikely that the 2.5- to 4-fold higher cell dose used for transplantation of 300 cGy recipients can account for the increased frequency of vector marking and the relatively greater fraction of integration sites associated with cancer genes. HSC represent ≈ 0.01% of mouse BM cells47
and transplantation of 2 × 106
transduced cells corresponds to transplantation of approximately 200 HSC. While the precise number of HSC after gene transfer is hard to estimate, as 5-FU treatment will enrich for HSC in marrow progenitors while ex vivo transduction can decrease their number, the number of HSC infused in our model appears to be considerable, given the number of clones actively contributing to hematopoiesis post-transplant. Moreover, HSC do not appear to be limiting as new clones continue to emerge after serial transplantation. Most importantly, the 2.5- to 4-fold fold increase in cell dose administered to the 300 cGy group may explain the relatively greater diversity of integration sites in this cohort, but it would not be predicted to alter the rate of gene transfer into the population. Therefore, the higher cell dose would not account for the higher frequency of vector-positive donor cells and of integration sites involving cancer-associated genes.
The small degree of overlap of RIS in serial transplants with those in primary recipients suggests that reconstitution of hematopoiesis involved activation of previously dormant vector-containing HSC. Unlike prior studies,11,12
we did not detect a further enrichment upon serial transplantation for RIS involving genes associated with cancer () or signal transduction (Fig. S3
). Approximately 10% of the RIS identified in our study are present in a database of 280 RIS identified in primary and secondary recipient mice in these studies,12
which also used SFFV-based vectors. These differences between laboratories may reflect differences in vectors or experimental conditions.
Despite the occurrence of 15% or more of vector insertions in or near cancer-associated genes, the incidence of leukemia/lymphoma was low in our study, even in serial transplants, a procedure that can promote vector insertion-related leukemic progression.28
. Only 4 malignant cases were identified, all in tertiary recipient mice; leukemic tissue was vector-positive in one case whereas two appeared to be host-derived. This is consistent with observations indicating that vector-induced leukemia is uncommon in the time frame of the mouse transplant model unless there are multiple insertions.28,48
It is difficult to compare the overall incidence of leukemia with other murine gene therapy studies, due to differences in retroviral vector backbones, transduction protocols, conditioning regimens and mouse strains. The incidence has varied significantly from study to study, and is further confounded by a variable incidence of vector-negative leukemia which has been reported to be as high as 6 out of 40 primary recipients in one study.49
This variability has been speculated to reflect effects of irradiation along with possible activation of endogenous retroviruses (see 49
In the current study, 5 of 184 independent provirus insertions in primary recipients occurred in or near Evi1
and two mice (A16 and A4) had evidence of expansion or dominance of an Evi1
-positive clone. Insertions in Evi1
are often over-represented in gamma-retrovirus-mediated gene transfer studies of murine hematopoietic cells either in vitro 50,51
Insertions in EVI1
or the adjacent MDS1
were also enriched in a non-human primate gene therapy study, accounting for 14 of 702 vector integration sites.13
activation has been implicated as a cooperating event in murine and human myeloid leukemias,52
we did not observe leukemia in either primary or serial transplant recipients harboring Evi1
vector in this study was also used in a recent clinical trial, where two patients exhibited myeloid expansion of clones with activating insertions involving MDS/EVI1
that accounted for more than 50% of granulopoiesis.4
Both patients subsequently developed MDS with Monosomy 7, involving a MDS/EVI1
-insertion positive clone.(M. Grez, personal communication) RIS recovered in our mouse transplant model using this vector were clearly more diverse, and domination of myelopoiesis with clones harboring integrations involving Evi1 was uncommon. The differences between these findings and the clinical trial could be related to many factors, including differences in the transduced cell population, the conditioning regimen and murine vs primate hematopoiesis, illustrating the challenges and limitations in predicting potential outcomes in the clinical setting using the murine model.
Finally, additional studies are required to determine if the findings noted with the SF71gp91phox
vector are applicable to other gamma-retroviral vectors or to self-inactivating lentiviral vectors, where insertions in or near cancer-associated genes appear to be much less frequent.9
Of note, in our previous studies using nonmyeloablative conditioning in combination with an MSCV-based vector,22–24
we did not observe an increased fraction of oxidase-corrected neutrophils in sub-myeloablated cohorts relative to recipients receiving high dose radiation, suggesting that the potent enhancer in SF71gp91phox,4
perhaps contributed to the biases seen in the current study.
In summary, our data confirm that certain integrations in HSCs and their progeny are over represented in long-term primary transplant recipients, and further suggest that this bias may be influenced by the marrow environment at the time of transplantation. Our data highlights the importance of modeling all aspects of a transplant approach in the murine system, as reduced intensity conditioning of a host for transplantation would have been predicted to result in less morbidity to the host, when in fact, in the context of gene replacement therapy in HSC, a potential deleterious effect was noted. The analysis of vector integration sites in the setting of partial ablation may thus be a useful approach to augment other assays aimed at examining the potential for vector-induced alterations in hematopoiesis.