ALBERT

Description: CGD is a monogenetic disorder that results in significant morbidity and mortality and is therefore a good candidate for gene therapy techniques. Genetic modification has been used successfully in mouse models of the disease and further, allogeneic bone marrow or peripheral blood transplantation has been shown to be curative in humans. The first clinical trial for CGD performed by Malech et al., (PNAS 1997) done without conditioning, resulted in marking levels of 0.1 to 0.01%. More recently with the use of busulfan 8mg/kg and an SFFV retrovirus, Ott et al., (Nature, 2006) achieved much higher (〉20%) peripheral blood levels of corrected cells. Subsequently there was an oligoclonal expansion of those cells in which probable activation of myeloid specific genes EVI1-MDS, SetBP1 or PRDM16 occurred due to vector insertion. While there was low production of superoxide on a per cell basis, there was a clinical benefit to these patients as their underlying infections resolved with the therapy. As such, we have initiated a clinical trial with the primary goal to treat infections in X-CGD patients with genetically modified cells. We used busulfan 10mg/kg over two days as a conditioning agent and a standard ex vivo transduction method using an amphotropic pseudotyped MFGS vector encoding only the gp91phox transgene, culturing the cells for 96 hours in retronectin coated bags and media supplemented with, SCF, Flt-3, IL-3, and MDGF. To date we have treated two patients. The first is a 28 yo male who presented with multiple staph aureus positive liver abscesses not amenable to resection or radio frequency ablation. He received 40x10e6 CD34+cells/kg after the busulfan and the transduction efficiency was 73% as measured from Day 4 of the culture. At day 13 post transplant, the patient had a marking level of 24% that subsequently decreased over time, but now remains at approximately 0.7–1% more than 9 months post treatment. The patient tolerated the treatment well and 6 months after his treatment, his liver abscesses were completely resolved. He remains on routine antibiotic prophylaxis. LAM PCR analysis in this patient has not shown any clonal dominance. We have found one insert in MDS-EVI1 out of 60 insertions found and sequenced, but none in SetBP1 or PRDM16. The second patient, a 31 year old male, was enrolled due to a fungal chest wall infection unresolved despite two years of combination antifungal treatment. The patient’s cells were transduced with the same vector as the first, but using a different production lot with a lower titre. The transduction efficiency was therefore lower with 41% gp91 positive cells at end of transduction. The patient received a total of 71x10e6 CD34+cells/kg and the initial marking was 5% at two weeks post infusion while the neutrophil count was still low but recovering. By day 21 however, the marking had dropped to less than 1%. Interestingly, there was a small positive fraction in the unstimulated control of the DHR, suggesting auto-activation of the cells. Hence given the timing of the drop and the presence of these autostimulated cells, we hypothesize that the patient has had an immune mediated reaction to the gp91phox expressing cells. It does not appear to be silencing as the realtime PCR data also suggests clearance. Studies are in progress to determine if this is a T or more likely, B cell mediated rejection. In the meantime, we will add an immunosuppressant peritransplant to avoid any possible immune mediated rejections in future patients.

Description: Key Points The genetic cause of SCID impacts on survival and immune reconstitution and should be considered in tailoring HCT for individual patients. Total and naive CD4+ cell counts in SCID patients 6 and 12 months post-HCT predict long-term survival and sustained immune reconstitution.

Description: Hematopoietic cells can be highly enriched for repopulating ability based upon efflux of the fluorescent Hoechst 33342 dye by sorting for side population (SP) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We sought to determine the effects of forced expression of the ABCG2 gene in hematopoietic stem cells in the nonhuman primate model, a model with proven relevance to human hematopoiesis. We cloned the full-length rhesus ABCG2 (rh-ABCG2) cDNA using a series of primers spanning the entire sequence designed using the published human sequence. Sequence homology was greater than 96%. The rh-ABCG2 gene was then introduced into an MFGS based retroviral vector pseudotyped with the RD114 envelope. Mobilized human peripheral blood CD34-positive cells were transduced with either rh-ABCG2 or human GP91-phox vector with no other payload. All transductions were initiated with 4 x10e5 cells using X-VIVO10/1%HSA/4mM/L L-glutamine supplemented with 100ng/ml_FLT3L, 100ng/ml SCF, 100ng/ml TPO and polybrene (5 ug/ml). RD114 vector was concentrated by ultracentrifugation (83,000g 90minutes 4°C). Gene transfer rates to CFU of greater than 80% were achieved using both vectors with similar gene transfer rate estimated by flow cytometry. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype, but showed significantly reduced growth compared to control (Day 8: cell counts 7.67+/− 2.54 vs. 17.83+/−6.64 x10e5 for ABCG2 and GP91-phox transduced cells, respectively p=0.0024, n=5). We then examined the engraftment of ABCG2-expressing stem and progenitor cells in the rhesus macaque autologous transplant model. GCSF/SCF mobilized PBPCs were collected from 2 animals and the CD34+ cells were divided and transduced with either vector and infused after lethal irradiation. In vivo marking levels post transplant measured in mononuclear cells and granulocytes from peripheral blood and bone marrow ranged initially from 0.5–4% by Realtime PCR, declined equally over time, and were similar between transduced fractions, with no discrepancy between bone marrow and peripheral blood marking. Furthermore, peripheral blood T cells, B cells and granulocytes expressed ABCG2 at levels predicted by vector copy number long term, and the differential of such cells within the SP gate matched that of the non-SP fraction demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus-ABCG2, support its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon hematopoiesis, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

Description: Chronic Granulomatous Disease (CGD) results from a mutation in the NADPH oxidase complex. As a result, patients are prone to recurrent infections and an increased risk of autoimmune disorders such as colitis. Currently, the only available cure is hematopoietic stem cell transplantation using a related or unrelated donor. In 2002, the NIH published their results using a nonmyeloablative regimen and sibling matched donors. Although the results were overall promising, there was still significant GvHD in older patients and a number of graft rejections in the younger patients. Further accrual was limited due to donor unavailability. In 2007, after establishing an agreement with the National Marrow Donor Program, we were able to initiate a protocol for patients with primary immunodeficiencies using an HLA matched unrelated donor. The goal of this protocol was to achieve engraftment in patients with CGD, including high risk patients due to the presence of an ongoing infection or inflammation, without increasing the rate of graft versus host disease. We therefore devised a novel conditioning regimen of Busulfan, Campath, and TBI along with sirolimus as the sole GVHD prophylaxis. To date we have transplanted 21 evaluable patients. Results are summarized below:AgeInfectionInflammationaGvHDcGvHDadditional cells?A&Wcause of death32XX1Nrefused dialysis25XGrade 1Y21XXGrade 1Y19X (colostomy)Grade 4XNinfection, GvHD of skin17XX2NEvan's/TRALI/GvHD17XNpulmonary hemorrhage17XGrade 2Y12XlimitedXY7XY8XY8X (colostomy)Grade 1Y8XXY6XX3NGvHD after 3rd transplant5XY4XY4XXGrade 2Y17Y11Grade 1Y10Y10Y8Y 1. Received peripheral blood stem cells from same donor after receiving bone marrow 2. Received cells after additional conditioning in the setting of Evan’s syndrome 3. Received additional cells with initial graft failure. Went on to a second then third transplant with a different conditioning regimen and different donor. Overall survival was 76%; however all deaths occurred in high risk patients and 2 of the 5 were unrelated to the initial transplant. Further, all surviving patients transplanted with high risk disease (11 of the 16) continue to have stable engraftment and had complete resolution of their inflammation and/or infection. This includes a patient with P40phox deficiency whose primary manifestation of CGD was colitis as well as a patient with an invasive fungal infection requiring emergency laminectomy 3 weeks prior to transplant. We have had limited GvHD to date and this occurred primarily in the high risk patients (6 of the 7). The most severe GvHD occurred in a patient given additional cells due possible poor engraftment and persistent thrombocytopenia. In retrospect, this may have been a sign of GvHD and not graft failure; however the result was severe GvHD of the skin and ultimately death from sepsis. Further, the only chronic GvHD (transient, now resolved) was also in a patient given additional cells for concerns of possible graft failure. Subsequently, the protocol was modified to no longer give additional unmanipulated cells and no graft failures or any severe GvHD has occurred in any of the subsequent patients. In general, patients tolerated the transplant well, although we did see a higher than expected rate of engraftment syndrome, again in the high risk patients only (5 of 21). Many patients needed only 1 or 2 transfusions of either platelets or red blood cells and 3 did not require any transfusions at all. We also transplanted two patients with CGD/McLeod’s, banking autologous blood prior to the transplant, and only one patient required any blood (1 autologous unit). Three patients did require multiple infusions due to prolonged time to engraftment or slow platelet recovery including the one patient to receive bone marrow as their initial donor product. For the one patient with late graft failure, there was autologous recovery. Thus in this single centre study we have transplanted 21 patients to date including 16 of those considered high risk using a novel non-myeloablative transplant regimen and an unrelated donor. We have had significantly lower rates of GvHD (33%) of which

Description: Chronic Granulomatous Disease is an inherited immunodeficiency resulting from a defect in one of 5 proteins necessary for the production of NADPH oxidase, an important component in managing infections by neutrophils. The most common form of the disease is X-linked due to mutations in the GP91 protein. Patients with CGD are thus prone to various infections from specific bacteria and/or fungi, but generally have normal viral responses. They are also prone to inflammatory diseases, including sarcoid, lupus, and colitis. Allogeneic hematopoieitic transplantation has been shown to be curative and will arrest or even reverse some of the inflammatory problems and is now more commonly used in patients with a history of inflammation or recurrent infections. In 2009, a new form of CGD was described resulting from a mutation in the P40 protein and to date only 2 patients have been described with this form of CGD. Unlike the other subtypes, these patients did not present with infection but in fact with severe colitis, making their diagnosis much more difficult. The role of P40 versus the other components of the NADPH oxidase does not necessarily explain this particular presentation as opposed to infection susceptibility; however assays such as DHR to test oxidase production can be misleading (both patients demonstrated only a partial reduction of oxidase production by DHR assay with normal sequencing for standard CGD subtypes) and may need to be performed specifically for a P40 dysfunction. We now report on the results of allogeneic transplant for two patients with P40 CGD. The first patient was referred to the National Institutes of Health (NIH) at the age of 9. His initial presentation was at an early age with diarrhea and perirectal abscesses. Notably, on his first EGD and colonoscopy he had evidence of granulomatous disease. He was eventually referred to a second institution for further testing and determined to have compound heterozygous mutations in the NCF4 genes with a frameshift mutation resulting in a premature stop codon in one allele and a missense mutation in the other. (Blood 114: 3309-3315, 2009) He had had a diverting ileostomy done at his referring institution as a result of his refractory colitis and was still requiring immunosuppression for his disease at the time of referral for transplant. He had no matched sibling donors and therefore underwent transplant using a 9 out of 10 matched unrelated donor. The second patient was a 17 year old male diagnosed at the age of 15, again with colitis and significant perirectal disease, with treatment including Interferon gamma, high dose corticosteroids, topical anti-inflammatories, azathioprine, and long-term antibiotics. His diagnosis of P40 CGD was made at the NIH where sequencing confirmed a compound heterozygous mutation of NCF4 with frameshift mutations in Exon 3 and Exon 9. His unaffected sister was a full match and was his donor. Both patients received busulfan (5mg/kg and 10mg/kg respectively) and Campath as part of their conditioning regimen with 300cGY of TBI also given for the MUD transplant. Sirolimus was used as the sole GvHD prophylaxis. The products were unmanipulated peripheral blood stem cell grafts (doses 8x10e6 and 10.4 x 10e6 CD34/kg respectively) and both patients had full myeloid engraftment by Day 30. The first patient had some grade 1 GvHD of the skin and GI tract initially, but is now more than 3 years post transplant and is considering revision to reconnect his ostomy with no evidence of GvHD, chronic or otherwise. The second patient is also doing well more than 1 year out, with no evidence of GvHD and has had complete resolution of his colitis off of his immunosuppression. Thus, allogeneic transplant with either a matched related or unrelated donor appears to be curative in patients with this rare form of CGD, leading to complete resolution of their inflammatory complications. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1329 Chronic granulomatous disease (CGD) is a congenital disorder resulting from decreased or absent oxidase production by neutrophils. As a result, patients with CGD are prone to bacterial and fungal infections and have a shortened life expectancy. Hematopoietic stem cell transplantation (HSCT) can be curative, and patients are increasingly referred for transplant due to an ongoing infection not amenable to cure using standard treatments. The use of granulocyte transfusions has been described in patients undergoing transplant with an underlying infection at the time of conditioning, but reports are limited to single cases. We describe here the effects of granulocyte transfusions in four patients undergoing either an HLA matched sibling or unrelated donor HSCT, using an alemtuzumab containing regimen and sirolimus as GVHD prophylaxis. Age of the patients was 6 to 25 (mean 11.5) yrs, and weight was 15.3 to 54 (mean 27.5) kg. Three of the patients had persistent Aspergillus infection involving the spine, lung, and/or brain despite long term combination antifungal therapy including a triazole, echinocandin and/or amphotericin. The fourth patient had an Actinomyces pneumonia progressive on combination antibacterial treatment. None of the patients had detectable HLA antibodies prior to the transfusions, and none received G-CSF post-transplant. The granulocytes were started on the anticipated day of neutropenia after conditioning and graft infusion and continued until evidence of graft recovery. Volunteer community donors underwent mobilization with dexamethasone 8 mg PO plus G-CSF 480 mcg SC, followed the next day by a 7-liter leukapheresis procedure (Spectra) using Tricitrasol/Hetastarch (Hespan) as the anticoagulant/sedimenting solution. Patients received a mean of 4.5 transfusions each, with three patients receiving biweekly transfusions and one receiving transfusions three times a week. The mean number of granulocytes per component transfused was 6.17 × 10e10 (range 4.0–9.12). This resulted in a mean of 2.78 × 10e9 granulocytes transfused per kg recipient weight (range 0.76–4.48). All patients tolerated the infusions well, without respiratory symptoms. The mean increase in absolute neutrophil count posttransfusion was 1.95 × 10e3/uL (range 0.5–4.61), although the timing of collection of the blood count samples was variable for each transfusion. Using a dihydrorhodamine (DHR) fluorescence based assay we were able to track the presence of oxidase positive cells to help differentiate transfused donor from residual host cells. DHR assays were performed before and after every transfusion in one patient. The number of DHR positive cells prior to infusion was 0–1.4% with a rise to 68.2% 24 hours after transfusion. 24.5% oxidase positive cells were still detectable almost 72 hours after transfusion, just prior to the next transfusion. One product required sedimentation for ABO incompatibility. The mean time to engraftment was 22 days and did not differ in patients with CGD receiving the same transplant regimen with (n=4) or without (n=6) peri-transplant granulocyte transfusions (30 days). Although all four patients had infectious processes not responding to standard antimicrobial therapy prior to transplant, there was no evidence of progressive infection during the period of neutropenia. Imaging studies of the brain, lung and/or spine suggested improvement in the infectious process in all four patients, although the timing of the scans made assessments inconclusive. Our prior experience with granulocyte transfusions in patients with CGD not undergoing transplant is that the cells do not persist in the circulation longer than 24 hours. Higher cell doses per kg in our four patients may have contributed to the sustained detection of transfused circulating cells, particularly as three of the patients weighed less than 21 kg. Peri-transplant immune suppression may also have facilitated the longer circulation time of the cells. We conclude that community-donor granulocyte transfusions are well tolerated in non-alloimmunized CGD patients undergoing HSCT, do not impact engraftment, are logistically feasible, and may provide a therapeutic bridge for patients with an underlying infection during a prescribed period of neutropenia, thus decreasing transplantation risk for such patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3744 Graft versus host disease (GVHD) is a serious complication of allogenic hematopoietic stem cell transplantation (HSCT) affecting primarily the liver, intestine, skin and lymphoid organs. T-regulatory cells (Tregs) are a subset of T cells that are characterized by CD4+CD25high and express the Foxp3 transcription factor. There is increasing recognition that Tregs likely are important in preventing development, reducing the severity and/or mediating resolution of GVHD. It has been reported that giving donor Tregs will suppress development of GVHD in a mouse model; however it is not clear whether the immunologic suppression by Tregs occurs in the target organs. Agonists of the Gs-coupled Adenosine A2A receptor (A2AR) agonists have been demonstrated to terminate inflammation and improve survival in solid organ transplant ischemia models. We previously have reported that the A2AR specific agonist, ATL146e, also can decrease the incidence and severity of GVHD as well as improve survival of mice in a GVHD transplant mouse model. In this same GVHD mouse model, ATL146e treatment increases the number of donor derived CD4+CD25highFoxp3+ Tregs. In order to further understand the role of the agonist in GVHD abrogation we performed studies looking at A2AR agonist mediated appearance of Tregs in the target organs affected by GVHD in this model. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2b] -〉 B6D2F1/J [BDF1, H-2b/d]) we can induce GVHD as manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells using 850cGy conditioning. We administered the A2AR specific agonists, ATL1223 or ATL370, or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14 days starting one day before the donor T cell infusion. Mice that received only the donor bone marrow transplant and not the additional donor T cells did not develop GVHD and served as an additional control. We collected ear and colon biopsies from the transplanted mice to assess these target organs (skin and gut) usually affected by GVHD in this model. Tissue was obtained, placed in 10% formalin and paraffin embedded and stained by immunohistochemistry (IHC) for intracellular FoxP3 by incubating overnight with 1:200 mouse anti-mouse/human/rat—FoxP3 IgG. The slides were washed and then incubated with anti-mouse IgG conjugated to Tetramethylrhodamine (TRITC). We then counted the number of Foxp3+ cells per high power field. We also examined whether specific agonist activation of A2AR can increase the number of Tregs in vitro by culturing CD4+ CD25− cells in the presence of either TGF-beta and/or the A2AR specific agonists. As in our previous studies with the specific A2AR agonist ATL146e, the agonists ATL1223 and ATL370 also were effective at preventing the acute GVHD syndrome of weight loss and mortality seen in the PBS treated controls. Treatment with these agonists resulted in an increase in CD4+CD25+FoxP3+ Tregs in the spleen compared to the PBS treated group at days 14 to 20 after HSCT. In addition we were able to document significant increases in the number of Foxp3 positive T lymphocytes infiltrating skin and colon compared to the PBS treated group. The number of Foxp3+ T cells per high power field was 14 ± 6 in the control and 64 ± 14 in the A2AR agonist treated mice (p = 0.0281). Finally from our in vitro data we also observed a 4.2 to 5.4 fold increase in the number of Foxp3+ Tregs in vitro after A2AR activation compared to TGF beta treatment only. Thus we have confirmed that the specific activation of A2AR inhibits acute GVHD through the increase of donor-derived CD4+ CD25+ FoxP3+ immunosuppressive T regulatory cells in vivo and in vitro. Furthermore, our histological observation suggests the possibility that increased Tregs by treatment with A2AR specific agonist is responsible for suppressing the immune response in GVHD target tissues. Therefore, our observation provides additional mechanistic basis for the anti-inflammatory capacity of A2AR agonist in acute GVHD. Additional studies are ongoing to further elucidate the mechanism by which A2AR specific agonist increases Tregs. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic stem cell (HSC) transplantation remains the only curative approach for sickle cell disease (SCD) patients, yet high risk of procedural toxicities and graft-versus-host disease limits this approach. We chose a low-dose radiation approach utilizing rapamycin based upon its unique ability to promote T cell tolerance. We tested this approach in vivo in a murine bone marrow transplantation model comparing a short course of conventional immunosuppression with cyclosporine to that with rapamycin, with long-term, high-level chimerism attained only in mice treated with rapamycin. We have now begun accrual to an IRB approved clinical trial testing this approach in SCD adults and report the results in our first five subjects. Protocol entry criteria include irreversible complications from sickle cell disease (such as prior strokes or pulmonary hypertension defined as a tricuspid-regurgitant jet velocity [TRV] ≥2.5 m/s), or reversible complications (such as frequent vaso-occlusive crises or acute chest syndrome) not ameliorated by a 6-month course of hydroxyurea. Conditioning was achieved with a single radiation dose of 300cGy, alemtuzumab (1mg/kg total), and oral rapamycin targeting trough levels between 10–20 ng/ml. All patients received unmanipulated mobilized peripheral blood progenitors obtained from an HLA-matched sibling. Assessment of donor chimerism was measured by microsatellite PCR of CD3 and CD14/15 positive white cells. Conditioning was well tolerated in all patients. Two patients inadvertently received only 200 cGy. CD34+ cell doses ranged from 5.72–10 × 10e6/kg, and CD3+ cell doses ranged from 1.93–5.35 × 10e8/kg. In 4 of the 5 patients in whom follow-up is sufficient, an early peak in myeloid chimerism was seen at 95–100% at day 28, with stabilization at 48–100% long-term. Lower level lymphoid chimerism was seen early, ranging from 3–23%, with stabilization at 5–47% long-term. Hemoglobin electrophoresis revealed complete replacement by donor type hemoglobin in all 4 patients by day 120, with amelioration of the phenotype allowing for therapeutic phlebotomy. Unfortunately, two patients have experienced recurrent sickle cell disease, though T cell responses to donor assessed by the mixed lymphocyte reaction revealed continued tolerance to donor with preserved T cell responses to third party antigens. These results argue against rejection and suggest competition by surviving endogenous HSCs as a cause of graft loss. Additionally, retransplant of one of the patients in relapse thus far using 400 cGy, alemtuzumab, and rapamycin has re-established full donor erythroid chimerism. Stable mixed chimerism persists out to 23 months in one patient and 10 months in another. Finally, no patient to date has developed any symptoms or signs of acute or chronic GVHD. Our results demonstrate the ability of allogeneic HSC transplantation using a relatively simple conditioning regimen to achieve mixed hematopoietic chimerism without the development of GVHD, suggest the importance of adequate hematopoietic suppression, and may allow extension to alternative donor transplantation.

Description: Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91phox, achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.