ALBERT

Description: Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. Nonmyeloablative conditioning regimens such as low-dose total body irradiation (LDTBI) can enhance competitive engraftment in the absence of a selective advantage for HSC. Granulocyte colony-stimulating factor (G-CSF) treatment of recipients prior to LDTBI has been shown to enhance engraftment of fresh bone marrow (BM) cells, but the underlying mechanisms and whether this combined approach can enhance long-term engraftment of genetically transduced donor cells have not been defined. We examined whether pre-treatment of recipient mice with G-CSF prior to LDTBI improves long-term donor engraftment of retroviral vector-transduced HSC in murine X-linked chronic granulomatous disease (X-CGD). In initial studies, we verified that pre-treatment of wild type mice with 4 ug G-CSF SC BID for 5 days prior to irradiation with either 160 cGy or 300 cGy significantly increased long-term engraftment of congenic fresh BM cells (see Table). Donor → Host Cell dose LDTBI (cGy) Donor chimerism + vs - G-CSF (Mean ±SD) C57BL/6J → PtrcaPep3b/Boy J 20 x 106 fresh BM 160 65.2±0.9% vs 43.4±3.5%, n=4 and 5 mice each; p=0.0002 (4 months after transplant) C57BL/6J-EGFP → C57BL/6J 2.5 x 106 fresh BM 300 55.25±4.5% vs 28.7±5.3%, n=4mice each; p=0.0002 (6 months after transplant) C57BL/6J male X-CGD → C57BL/6J female X-CGD 5 x 106 transduced BM 300 51.0±13.1% vs 30.4±7.8%, n=5mice each; p=0.02 (3 months after transplant) We next investigated 2 potential mechanisms by which G-CSF prior to LDTBI might enhance long-term engraftment of HSC. First, analysis of host BM 20 hours after transplantation showed a significantly lower frequency of homed donor HSC in G-CSF+160cGy treated mice compared to 160 cGy-conditioned mice (1 ± 0.13% vs 3.5 ± 0.7% in one experiment, and 3.4 ± 0.8% vs 9.5 ± 2.3% in another experiment, n= 20 mice). Second, in a competitive repopulation assay, we observed a substantial decrease in marrow long-term repopulating ability (LTRA) in mice treated with G-CSF + 160 cGy compared to 160 cGy alone (10.5% vs 48.6%, n= 5 mice in each group; p= 0.0002). These results suggest that administration of G-CSF prior to LDTBI does not increase initial homing of donor cells but further impairs LTRA of recipient BM compared to LDTBI alone. In the context of gene therapy, we transduced male X-CGD BM with MSCV-gp91Neo vector and infused these transduced cells into female X-CGD mice treated ± G-CSF + 300 cGy. Donor cell engraftment, measured by FISH for Y chromosome, at 3 months post-transplant was significantly increased in recipients pretreated with G-CSF (see Table). NADPH oxidase activity (by 123-dihydrorhodamine assay) was reconstituted in 9.75 ± 5.5% of neutrophils compared to 6.6 ± 2.5% without G-CSF pretreatment. The calculated fraction of donor cells with oxidase activity was similar in both groups (18.8 ± 6.6% vs 21.4 ± 11%, respectively) (p= 0.67). These results suggest that the use of G-CSF prior LDTBI provides a competitive advantage for long-term engraftment by decreasing LTRA in the host and may be a useful strategy to increase engraftment of fresh and retroviral transduced donor BMC.

Description: Abstract 3759 For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures: No relevant conflicts of interest to declare.

Description: X-linked chronic granulomatous disease is an inherited disorder of innate immunity in which neutrophils are unable to generate microbicidal oxidants due to mutations in the gene encoding the gp91phox subunit of the NADPH oxidase. Genetic blood diseases can be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. In the absence of a selective advantage following genetic correction, marrow conditioning is required to facilitate engraftment. Using a murine model of X-CGD, we examined the efficacy of 300 cGy as conditioning prior to transplantation of HSC transduced with a monocistronic gammaretroviral vector, SF71gp91phox, in which human gp91phox expression is driven by the SFFV LTR. Although packaged with an ecotropic envelope for this study, this is the same vector as used in an ongoing clinical trial for X-CGD, where two reported patients showed an unexpected increase in the fraction of oxidase-corrected neutrophils, which appeared to reflect expansion of gene-modified myeloid cells due to insertional activation of adjacent loci (Ott et al, Nat Med, 2006). We compared donor engraftment and reconstitution of NADPH oxidase activity in female X-CGD mice conditioned with either 300 cGy or ablative irradiation (IR) (950–1100 cGy) prior to transplantation of transduced male marrow (5–8 × 106 and 2 × 106 cells, respectively). Donor chimerism in the 300 cGy group was 67 ± 14% by six months (mean ± SD, from two independent experiments), as monitored by FISH for the Y chromosome. The fraction of oxidase-positive cells in mice conditioned with ablative IR was 41 ± 11% and 18 ± 5% at 6 months in the two experiments, and was stable after 2 months post-transplant. In contrast, the fraction of oxidase-positive donor-derived neutrophils was higher in many 300 cGy-conditioned recipients; in the first experiment, 100% of donor neutrophils were oxidase-positive in 4 recipients at six months, and in the second, oxidase positive donor neutrophils increased to 100% in 1 of 8 recipients. The number of vector integrations in primary recipient spleen DNA was similar (≈2) for both conditioning regimens. However, the fraction of vector-marked secondary CFU-S derived from transplanted male cells was higher for mice receiving 300 cGy compared to those receiving ablative IR (vector-positive CFU-S 87 ± 19% vs 36 ± 20%, respectively; from 6–7 primary recipients for each regimen; p 〈 0.001). There has been no evidence of leukemic transformation in a total of 24 primary, 39 secondary, and 24 tertiary recipients (majority followed for ≥6 months post transplant). Ongoing studies include analysis of proviral insertion sites. In summary, 300 cGy is effective conditioning for engraftment of transduced marrow in a murine model of X-CGD. Our previous studies suggest that submyeloablative IR results in a more competitive environment for engraftment of donor cells cultured ex vivo for retroviral transduction (Goebel, et al Exp Hem, 2002). Analysis of proviral marking frequency in secondary CFU-S in the current study suggests that vector integration may confer a selection advantage for engraftment of long-term repopulating cells in primary recipients conditioned with 300 cGy, associated with a higher frequency of oxidase-corrected neutrophils.

Description: The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system. We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting. Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens. Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells. Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method. Disclosures No relevant conflicts of interest to declare.

Description: X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of innate immunity in which neutrophils lack the superoxide-generating NADPH oxidase, resulting in recurrent pyogenic infections. Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. However, in the absence of a selective advantage for HSC following genetic correction, marrow conditioning is required to enhance engraftment. The current study examined the efficacy of 300 cGy as submyeloablative conditioning using a murine model of X-CGD. We compared reconstitution of peripheral blood neutrophil NADPH oxidase activity in female recipients conditioned with either 300 cGy or ablative irradiation prior to transplantation of male bone marrow transduced with a monocistronic retroviral vector for expression of gp91phox, the product of the X-CGD gene, in the SFFV (spleen focus forming virus) backbone. Transduced cells were transplanted into each of 6 mice receiving 300 cGy (8 x 106 cells per recipient) and also into each of five 1100 cGy-irradiated mice (2 x 106 cells per recipient). Chimerism for donor cells in the 300 cGy group ranged from 40-75 % at 6 months (mean ± SD 63 ± 16%), as monitored by FISH for the Y chromosome. The fraction of oxidase-positive cells in the 1100 cGy group at 6 months was 41 ± 11%, and was stable in this range after 2 months post-transplant. Unexpectedly, the fraction of oxidase-positive donor neutrophils was higher in recipients conditioned with 300 cGy, which increased from 44 ± 17% at four months (N=6) to 100% (N=4) at six months, and was 61, 63, and 100% in 3 mice available for study at nine months. Two mice in each group were sacrificed at five months for analysis of vector copy number in spleen DNA, which was 2–3 in both the 300 and 1100 cGy-irradiated groups. In a second experiment, in 1 of 8 mice conditioned with 300 cGy prior to transplantation of 5 x 106 SFFV-91 transduced cells, the frequency of oxidase positive donor neutrophils was 75% at 5 months compared to 19 ± 6% oxidase-positive donor neutrophils in the other 7 mice and in the group conditioned with 950 cGy (18 ± 5%). Ongoing studies include analysis of provirus marking and insertion sites in secondary and tertiary CFU-S. There has been no evidence of abnormal hematopoiesis in primary and secondary transplant recipients. In summary, 300 cGy is effective conditioning for engraftment of transduced marrow in a murine model of X-CGD, and some recipients had an unexpectedly higher fraction of oxidase-corrected donor neutrophils compared to cohorts conditioned with ablative irradiation. Our previous studies suggest that recipients conditioned with submyeloablative irradiation provide a more competitive environment for engraftment of donor cells undergoing the retroviral transduction procedure (Goebel, et al Exp Hem30, 1432, 2002). We thus postulate that a higher frequency of gene-corrected donor neutrophils in 300 cGy-conditioned murine recipients might reflect a selective advantage for proviral integration at loci that are active in myeloid cells.