458. Reversal of Canine Leukocyte Adhesion Deficiency by Retroviral-Vector Mediated Gene Therapy with Non-Myeloablative Conditioning

ADVANCES IN HEMATOLOGIC GENE THERAPY 456. HOXB4 Overexpression Expands ShortTerm Repopulating Cells in Nonhuman Primates and Dogs Xiao-Bing Zhang,1 Laura J. Peterson,1 Alixandra A. Knapp,1 Brian C. Beard,1 R. Keith Humphries,2 Hans-Peter Kiem.1 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of British Columbia, Vancouver, BC, Canada. Human HOXB4 overexpression is able to extensively expand hematopoietic repopulating stem cells in murine models. Here we examined the effect of HOXB4 in two commonly used large-animal models using competitive repopulation assay. Three macaques and five dogs were transplanted with peripheral blood CD34+ cells transduced with a HOXB4/GFP or a control YFP gammaretroviral vector after lethal irradiation. Two dogs and one monkey died from transplant related complications. Follow-ups for the other three dogs and two monkeys are eight months and nine months, respectively. For all animals pretransplant gene transfer efficiencies in CD34+ cells were similar between HOXB4/GFP and YFP transduced cells: 45% (range 36-55%) and 38% (range 36- 40%) in macaque cells, and 48% (range 23-69%) and 51% (range 31-62%) in dog cells. In all 3 monkeys the initial gene transfer levels in vivo one week after transplantation were higher with the control YFP vector (average, 32% vs 25%). A similar trend was observed for three of the five dogs (average, 21% vs 11% for YFP vs GFP). In all the animals, dogs and monkeys, we observed a dramatic increase in HOXB4/ GFP marked cells from 5-30% to 10-62% during the early engraftment period resulting in a significant difference between HOXB4/GFP and YFP marked granulocytes. The peak ratio between HOXB4overexpressing to YFP-expressing in the three surviving dogs was approximately four at 5-6 weeks post-transplant. In the monkeys the peak ratio was up to ten at 5 weeks post-transplant. Interestingly, in all the dogs the HOXB4/GFP transduced cells decreased to within 2 fold of the control vector by week 15. Taqman PCR analysis of samples at the latest time point in all animals confirmed that the decrease in gene-marked cells was not due to transgene silencing. Subset analysis showed that the ratios of HOXB4-overexpressing cells to YFP-expressing cells were obviously higher for granulocytes than for lymphocytes in macaques and dogs, indicating that HOXB4 overexpression may inhibit lymphoid differentiation or/and promote higher level expansion of myeloid progenitors than lymphoid progenitors. In conclusion, our preclinical studies show that HOXB4 overexpression in CD34+ cells can expand hematopoietic cells that boost short-term engraftment.

457. The Benefits of CH-296 during Ex Vivo Gene Transfer Go beyond the Ability to CoLocalize the Retroviral Particle and Target Cell Mo A. Dao,1 Jan A. Nolta.1 Medicine/Oncology/Stem Cell Biology, Washington University, St Louis, MO.

1

Successful gene therapy depends on at least two parameters: introduction of the corrective gene into primitive hematopoietic progenitor cells (HPC) and maintenance of the long-term engraftment potential of these ex vivo manipulated progenitors posttransplantation. The use of the recombinant fibronectin fragment, CH-296, increases retroviral gene transfer by co-localizing the retroviral particle and the target cell which expresses VLA-4 and VLA-5 integrins. In a xenotransplantation model, our lab previously demonstrated the maintenance of long-term reconstituting hematopoietic progenitors during ex vivo gene transfer either on a pre-established irradiated bone marrow stromal monolayer or CH296-coated plates. In contrast, HPC cultured on BSA-coated plates in the presence of IL-3, IL-6, and SCF maintained short- but not Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

long-term engraftment potential. Here, we compared the molecular changes that occur in bone marrow and cord blood HPC cultured on CH-296 vs. BSA-coated plates in the presence of cytokines. Bone marrow HPC were cultured in the presence of IL-3, IL-6, and SCF for 48 hours. Subcellular fractionation followed by immunoblotting showed an induction of p21Cip1 protein in the cytoplasmic compartment of HPC cultured on BSA-coated plates. Since p21Cip1 is upregulated during monocytic differentiation, we hypothesized that lack of integrin engagement in the presence of cytokines might induce the loss of HPC primitive characteristics. Confirming our hypothesis, we observed a loss of c-myb, CD34, and GATA-2 expression in HPC maintained in suspension. In addition, we observed changes in MAPK phosphorylation and localization in HPC cultured on fibronectin, as compared to BSA-coated plates. Extrapolating the same hypothesis to cordblood derived HPCs, we observed consistently higher levels of reactive oxygen species (ROS) in cordblood HPC on BSA-coated plates, as compared to cells on CH-296. Aside from regulating cell differentiation, high levels of ROS have been shown to induce apoptosis. Consistent with this, we documented lower levels of phosphorylated cytoplasmic AKT in HPC on BSA-coated plates, as compared to cells on CH-296 plates. Based on these preliminary observations, we hypothesize that the suppression of differentiation markers such as p21Cip1 and apoptotic inducers such as intracellular reactive oxygen species, the maintenance of transcription factors such as GATA-2 and cMyb, and the enhanced phosphorylation of the pro-survival regulator AKT in HPCs during ex vivo culture on CH-296 may all contribute to the ability of these cells to achieve long term engraftment following transplantation into sublethally irradiated immunodeficient mice. We therefore propose that inclusion of CH-296 during ex vivo culture of HPC may be important even during non-retroviral mediated gene transfer in order to maintain HPC engraftment potential.

458. Reversal of Canine Leukocyte Adhesion Deficiency by Retroviral-Vector Mediated Gene Therapy with Non-Myeloablative Conditioning Thomas R. Bauer, Jr.,1 Laura M. Tuschong,1 Mehreen Hai,1 Yuchen Gu,1 Robert A. Sokolic,1 Tanya Burkholder,2 John D. Bacher,2 Dennis D. Hickstein.1 1 Experimental Transplantation and Immunology Branch, National Cancer Institute, NIH, Bethesda, MD; 2Office of Research Services, Division of Veterinary Resources, NIH, Bethesda, MD. Children with leukocyte adhesion deficiency or LAD suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD is due to heterogenous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency or CLAD, like children with LAD, also experience severe bacterial infections and typically die within the first few months of life. CLAD represents a disease-specific, large animal model for testing new therapeutic approaches for the human disease LAD. We tested a retroviral-vector mediated gene therapy approach in CLAD using a non-myeloablative conditioning regimen. Seven CLAD dogs received autologous, CD34+ gene-corrected cells following 200 cGy total body irradiation (TBI). CLAD CD34+ cells were prestimulated overnight with growth factors cIL-6, cSCF, hFlt3-L, and hTPO, then incubated with retroviral vector PG13/MSCV-cCD18 over 48 hours on recombinant fibronectin. Transduction of the CLAD CD34+ cells ranged from 15 to 33%. Following transduction, cells were re-infused (0.3 to 1.8 x 106 CD18+ cells per kg) after 200 cGy TBI. One group of 4 dogs received post-transplant immunosuppression consisting of cyclosporine and mycophenolate mofetil. A second cohort group of 3 dogs received no post-transplant immunosuppression. Peripheral blood samples were analyzed by flow cytometry for CD18 expression. At a mean time of 6 months S177

ADVANCES IN HEMATOLOGIC GENE THERAPY post-gene transfer, the CD18+ gene-corrected leukocyte frequency in the peripheral blood ranged from 0.044% to a high of 3.15%. Five of the 7 CLAD dogs (3 with immunosuppression, 2 without immunosuppression) receiving CD18+ gene corrected cells have had significant improvement of their CLAD disease and are alive and well at 9 to13 months of age. These results contrast markedly with those seen in untreated CLAD dogs that die or are euthanized within the first few months of life due to intractable infection. Our studies indicate that a non-myeloablative regimen of 200 cGy TBI regimen facilitates the engraftment of sufficient autologous, CD18gene corrected cells to correct the disease phenotype in CLAD, and that post-transplant immunosuppression is not required for the persistence of CD18 gene-corrected cells. These results provide support for the use of a non-myeloablative conditioning regimen prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.

459. Long-Term Treatment of Canine Cyclic Neutropenia by G-CSF-Lentivirus William Osborne,1 Margaret Brzezinski,1 Ofer Yanay,1 Lanaya Waldon,1 Jeffrey Christensen,2 Denny Liggitt,3 David Dale.2 1 Pediatrics, University of Washington; 2Medicine; 3Comparative Medicine, Seattle, WA. Cyclic neutropenia (CN) occurs both in man and grey collie dogs and is characterized by recurrent periods of neutropenia leading to bacterial infections and shortened life expectancy. Daily rG-CSF provides therapy for patients and dogs. To treat CN by gene therapy we constructed a VSV-G pseudotyped lentivirus encoding canine G-CSF cDNA regulated by CMV promoter, cPPT from HIV 1 and PRE from human hepatitis B virus. At 7 weeks of age when an affected grey collie dog was weaned, serial blood counts were monitored. These data showed the dog’s absolute neutrophil counts (ANC) were cycling and observations over 4 cycles established a 12 day periodicity. The ANC mean±SD during nadirs and peaks were 560±787 cells/µl and 10,760±1,120 cells/µl respectively and overall counts were 5,240±4,790 cells/µl. Having established that neutrophils were cycling, recombinant canine G-CSF was administered subcutaneously at a dose of 1.5 µg/kg per weekday for two months. Although ANC continued to cycle, we observed significant increases in neutrophils both at the nadirs and the peaks of cycles (p<0.0001) with mean values of 4,860±2,670 cells/µl and 28,870±9,830 cells/µl respectively. Over this period the mean count was 17,820±11,100 cells/µl. After demonstrating that rG-CSF elevated neutrophil production cytokine administration was stopped and we administered IM 2x109 infectious units(IU) of G-CSFlentivirus. The dog showed no ill effects to the virus injections. Serial monitoring of blood cells showed elevated ANC for over 17 months with mean value of 29,230±12,930 cells/µl (n=217) that was significantly increased over both no treatment and rG-CSF treatment (p<0.0001). The increases in ANC from recombinant GCSF and lentivirus administration were associated with absence of clinical signs of infection and fever. The ability to readminister lentivirus is important. To evaluate this we administered lentivirus encoding rat G-CSF cDNA under the control of CMV promoter (1-2x107 IU, IM) to four rats and achieved significantly elevated neutrophil counts for up to 420 days (treated and controls; 4,835±930 and 1,890±570 ANC/µl, respectively, p<0.0001). On day 340 three rats that received GCSF-lentivirus were administered lentivirus encoding rat EPO cDNA (108 IU, IM). After three weeks rats receiving EPO-lentivirus showed significantly elevated % hematocrits (HCT) over controls (61.5%±4.2; vs 48.2%±1.9; p<0.001) and most importantly, the elevated HCT levels in naïve control rats were not significantly different to that observed in rats previously treated with G-CSFlentivirus. Administration of EPO-lentivirus did not affect neutrophil S178

counts of rats previously treated with G-CSF-lentivirus. Thus, sequential administration of G-CSF and EPO lentivirus both provided sustained transgene expression. We have now treated two affected dogs with single administrations of G-CSF-lentivirus that achieved therapeutic levels of neutrophil production for over 17 months. In rats lentivirus readministration provided high-level transgene expression. These data support lentivirus-mediated G-CSF delivery for the long-term treatment of patients with cyclic neutropenia.

460. Transplantation of Human Aldehyde Dehydrogenase Expressing Cells Leads to Widespread Tissue Distribution of Donor Cells in the Pancreas and Liver of NOD/SCID/MPSVII Mice David A. Hess,1 Timothy P. Craft,1 Louisa Wirthlin,1 Phillip E. Herrbrich,1 Alex A. Hofling,1 Mark S. Sands,1 Jan A. Nolta.1 1 Internal Medicine, Division of Oncology, Washington University School of Medicine, St Louis, MO. Beta-glucuronidase (GUSB) is a lysosomal enzyme expressed in virtually all cell types. Transplantation of GUSB-expressing human hematopoietic stem cells (HSC) into the GUSB-deficient NOD/ SCID/MPSVII mouse allows for the detection of xenotransplanted cells in hematopoietic and non-hematopoietic tissues, without reliance on the expression of human-specific cell surface markers or in situ hybridization (Hoffing, 2003). We are currently using the NOD/ SCID/MPSVII model to investigate the transfer of the GUSB gene from human cells into murine cells of GUSB deficient tissues. We recently characterized a novel population of reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin) and selection of cells with high aldehyde dehydrogenase (ALDH) activity (Hess, 2004). These ALDHhiLin- cells demonstrate robust hematopoietic engraftment in the NOD/SCID model and contain a subpopulation of cells (ALDHhiCD133+) that may not be fully restricted to hematopoietic lineages. Here, we have used the NOD/ SCID/MPSVII model to study the tissue distribution of ALDHhiLincells in multiple organs. Tail vein injection of 2x105 ALDHhiLincells into sub-lethally irradiated (300cGy) NOD/SCID/MPSVII mice (n=6) demonstrated hematopoietic chimerism in the BM (70.5±15.1%), spleen (7.0±2.8%) and peripheral blood (17.0±10.7%). In contrast, injection of 106 ALDHloLin- cells (n=5) did not produce consistent human cell engraftment. By using GUSBspecific histochemical staining, significant human engraftment was also detected in the non-hematopoietic (liver, pancreas, kidney, lung, heart, and brain) tissues of mice transplanted with ALDHhiLincells. GUSB activity was co-localized with CD45 expression in the BM of mice transplanted with ALDHhiLin- cells. However, flow cytometric analysis of liver tissue revealed a discrepancy between engraftment detected by a fluorescent GUSB substrate, compared to human CD45 cell surface expression. Histochemical staining confirmed the presence of GUSB+ cells that did not express human CD45, and uncovered GUSB expressing cells with typical hepatocyte morphology. These hepatocyte-like GUSB+ cells were also present after the transplantation of the ALDHhiCD133+Lin- subpopulation (n=4). In the pancreas, GUSB+ donor cells were localized in ductal regions and surrounding recipient islets. The ALDHhiLin- UCB cells demonstrated a previously uncharacterized high level of engraftment in non-hematopoietic organs of NOD/SCID MPSVII mice. We are currently investigating cellular fusion and nuclear reprogramming as a putative mechanism for the production of nonhematopoietic cells expressing donor cell-derived GUSB. This gene transfer potentially occurs through spontaneous fusion as previously demonstrated in liver (Willenbring, 2004) and muscle (Camargo, 2004). This phenomenon may be further exploited to deliver target genes into various tissues for the treatment of genetic abnormalities. Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy