ALBERT

Description: Children with the severe deficiency phenotype of leukocyte adhesion deficiency (LAD-1) suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD-1 is caused by heterogeneous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency (CLAD), like children with severe deficiency LAD-1, experience severe bacterial infections, and typically die within the first few months of life from infection. CLAD represents a disease-specific, large animal model for evaluating new therapeutic approaches for the human disease LAD. In these studies, we tested a retroviral-vector mediated gene therapy approach in CLAD. Autologous CLAD CD34+ bone marrow hematopoietic stem cells were pre-stimulated 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 was measured by flow cytometry for CD18+ cells and ranged from 11% to 21%. The transduced cells were re-infused (0.26 − 1.49 x 106 CD18+ cells / kg) into the dogs following the administration of two different non-myeloablative conditioning regimens: 5 CLAD dogs received autologous, gene-corrected CD34+ cells following 200 cGy total body irradiation (TBI) and 2 CLAD dogs received autologous, gene-corrected CD34+ cells following 10 mg/kg busulfan. Peripheral blood samples were analyzed by flow cytometry for CD18 expression following the re-infusion of the transduced CD34+ cells. The frequency of CD18+ gene-corrected leukocytes in the peripheral blood ranged from 0.04% to a high of 4.44% at 6 – 11 months post-gene transfer. Two of the five dogs in the first group and one of the two dogs in the second group that received CD18+ gene-corrected cells are alive and well on no prophylactic treatment at 9 – 14 months of age. Of note, the CLAD dog receiving busulfan conditioning has the highest level of CD18+ gene-corrected cells (4.44% at 6 months post-infusion), with the levels increasing at monthly intervals since the second month following re-infusion. 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. These studies indicate that a clinically applicable non-myeloablative regimen of either 200 cGy TBI or 10 mg/kg busulfan facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the lethal disease phenotype in CLAD. No evidence of monoclonality has been detected by LAM-PCR in any of the dogs with therapeutic levels of gene-corrected cells. In future studies we will optimize the transduction protocol in order to increase the number of CD34+ gene-corrected cells for infusion, as well as closely monitor the gene-corrected animals for any evidence of insertional mutagenesis or other complications related to the therapy. Together, these findings support the use of either of two clinically applicable, non-myeloablative conditioning regimens prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.

Description: Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.

Description: Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

Description: Leukocyte adhesion deficiency (LAD), a genetic disease caused by defects in the integrin CD18, results in a clinical syndrome in which the affected children suffer severe recurrent infections due to the inability of their leukocytes to adhere to endothelium and migrate to sites of infection. Approximately 75% of children with the severe deficiency phenotype of LAD die by two years of age. Allogeneic hematopoietic stem cell transplant (HSCT) preceded by a myeloablative conditioning regimen corrects the LAD phenotype, however this treatment carries significant regimen-related toxicity and mortality. Full donor chimerism after myeloablation is commonly accompanied by graft-vs.-host disease (GvHD). We utilized an animal model of LAD, canine leukocyte adhesion deficiency (CLAD), to test new transplant regimens for LAD. CLAD is due to a point mutation in canine CD18, and the phenotype closely resembles the severe deficiency phenotype of LAD. We have previously shown that treatment with 200 cGy total body irradiation (TBI), matched sibling HSCT, and immunosuppression with cyclosporine A (CsA) and mycophenolate mofetil (MMF) corrects the CLAD phenotype. Because TBI in children is associated with growth suppression, sterility and increased risk of cancer, translation of this approach to humans will be limited by concern for toxicity. To develop a non-TBI conditioning regimen for LAD transplants, we tested single-agent busulfan. CLAD pups were identified by absence of CD18 on leukocytes by fluorescence-activated cell sorting (FACS). Three dogs that lacked a DLA-matched littermate donor died at 2, 4 and 6 months of age, respectively. Three CLAD pups were given 10 mg/kg busulfan IV followed 2 days later by 6-17x10^6 CD34+ cells/kg from matched sibling donors and immunosuppression with CsA/MMF. Chimerism was assessed by CD18 FACS. Dogs were observed for evidence of infection, regimen-related toxicity, graft-versus-host disease and overall health. All three dogs engrafted, and two animals maintained CD18+ leukocyte levels of 17.0% and 19.0% at 16 and 4 months post-transplant respectively. In both dogs, blood counts are normal with no signs of CLAD. These dogs are clinically and behaviorally indistinguishable from their healthy littermates. The third dog has had decreasing CD18+ cells and at 12 months post-transplant has 0.2% CD18+ donor leukocytes. Despite requiring two courses of parenteral antibiotics since stopping immunosuppression, this dog has considerably exceeded the life expectancy of non-transplanted CLAD pups. Thus, two CLAD dogs have had complete correction of the phenotype, and the third has had partial correction. There has been no need for transfusion and no other regimen-related toxicity. Post-transplant fevers were easily managed with no cases of refractory infection. There has been no GvHD. Our data show that a regimen using a single, non-myeloablative dose of busulfan can lead to long-term engraftment, stable mixed chimerism and reversal of CLAD phenotype with minimal toxicity. This treatment may provide LAD patients with matched sibling donors with an alternative to either myeloablation with full donor chimerism and risk of GvHD or non-myeloabaltive transplant using TBI with its attendant complications.

Description: Canine leukocyte adhesion deficiency (CLAD) represents a large-animal model for the human disease leukocyte adhesion deficiency, type 1 (LAD-1). Both CLAD and LAD are characterized by life-threatening bacterial infections and are due to mutations in the leukocyte integrin CD18. We recently reported successful gene therapy in six dogs with CLAD following infusion of autologous CD34+ bone marrow hematopoietic stem cells transduced with the retroviral vector PG13/MSCV-cCD18. The level of CD18+ gene corrected leukocytes ranged from 1.3% to 8.4% at one year post-transplant in the six CLAD dogs. We continue to monitor four of the six dogs for long-term follow-up. To date no gene transfer-related adverse events have been observed in any of the dogs. However, verification of polyclonality of insertion sites in the gene-corrected cells and characterization of the genomic sites of retroviral integration remain critical safety issues. To this end, we have demonstrated the polyclonality of contributing retrovirus-transduced cells in all six dogs using linear-amplification mediated PCR (LAM-PCR) with DNA obtained from peripheral blood leukocytes up to 2 years following infusion of gene-corrected cells. Multiple clones were shown to contribute to hematopoiesis; no predominant clones emerged over time. Retroviral insertion sites in the six dogs were identified using ligation-mediated PCR (LM-PCR) followed by sequencing of the genomic sequence adjacent to the integrated viral 3′ LTR. Mapping of insertion sites was facilitated by the recent completion of the annotated dog genome sequence. To date, we have identified 341 unique retroviral insertion sites. Analysis of these insertion sites indicated that 46% of the sites are located within gene-coding regions of the genome (introns or exons). Another 35% of the sites were located within 50 kb of the transcription start site of a gene-coding sequence. These observations match previous reports of preferential integration of MLV retroviral vectors near coding sequences. The insertion sites were well-distributed across all chromosomal locations, with no evidence for insertional hotspots or preferential growth of clones containing common insertion sites: only six pairs of insertion sites were located within 5 kb of each other. Further analysis was performed on a sub-group of 294 genes that had insertion sites located either in them or within 50 kb of the transcription start site. 19 of these genes (6.5%) were present in either or both of two cancer gene databases (Sanger Institute Cancer Gene Census Table, Mouse Retrovirus Tagged Cancer Gene Database). Functional annotation of the 294 genes revealed some groups of interest: 17% of the genes were involved in phosphorylation, 4% were proto-oncogenes, 12% encoded zinc-finger proteins, 23% were involved in signal transduction, 2% encoded transforming proteins, 5% were involved in cell proliferation, 8% were involved in transcriptional regulation and 4% encoded protein kinases. We are continuing to sequence and characterize a large number of retroviral insertion sites from the gene-corrected CLAD dogs. Analysis of these insertion sites should provide considerable insight into the risk of genotoxicity through insertional mutagenesis in a disease-specific large-animal model of a human hematopoietic disease.

Description: Leukocyte adhesion deficiency-1 (LAD-1) is a genetic immunodeficiency disease characterized by recurrent, life threatening bacterial infections. The phenotype of LAD-1 stems from the inability of leukocytes from affected children to adhere and migrate to sites of infection as a result of mutations in the leukocyte integrin CD18. Canine leukocyte adhesion deficiency (CLAD) represents the homologue of the severe phenotype of LAD-1 in humans. In previous studies we have demonstrated that less than 500 CD18+ neutrophils/μl following non-myeloablative stem cell transplantation from matched littermate donors resulted in reversal of the CLAD disease phenotype. In this report, we describe two CLAD dogs (D128 and D144) with donor microchimerism and improvement from a severe to a moderate clinical phenotype of CLAD following matched littermate hematopoietic stem cell transplant. Prior to transplant both dogs displayed the hallmarks of CLAD phenotype with fever, severe infections, leukocytosis and were following the natural history of CLAD, which invariably leads to death by 6 months of age. Both dogs are maintained on only prophylactic antibiotics and are alive more than two years post-transplant. The calculated level of CD18+ donor neutrophils was approximately 100 CD18+ neutrophils/μl in dog D128 and 50 CD18+ neutrophils/μl in dog D144, respectively. To determine how this persistently low level of CD18+ neutrophils/μl measured in the peripheral blood resulted in the moderation of the severe disease phenotype of CLAD, we assessed the level of CD18+ leukocytes in mucosal tissues. Mucosal tissues have a constant bacterial presence that is kept under control in part by a constant influx of neutrophils from surrounding periodontal tissues. The emigration of neutrophils from the circulation is a critical step during immune surveillance and inflammatory reactions, and is governed by a coordinated interaction involving a spectrum of adhesion and signal molecules. The oral rinse assay enables a non-invasive, in vivo measurement of neutrophil migration into tissues. Both dogs displayed evidence of selective extravasation of CD18+ donor cells into extravascular site as shown by FACS analysis with eleven-fold (D128) and five-fold (D144) more donor-derived CD18+ leukocytes present in saliva compared to the peripheral blood. The percentage of donor chimerism was confirmed using DNA microsatellite markers that distinguished donor from host. These results indicate that a level of less than 100 CD18+ neutrophils/μl in peripheral blood improves the CLAD phenotype from a severe to a moderate clinical phenotype. Moreover, measurement of neutrophils in peripheral blood alone fails to take into consideration of the total number of CD18+ donor derived cells in the recipient CLAD animals post-transplant. These results suggest that measurement of donor-derived CD18+ neutrophils in the tissue in this disease provides a more accurate assessment of the clinical effect than the measurement of CD18+ leukocytes in the peripheral blood and they support the treatment of CLAD by gene therapy, where a low number of gene corrected cells is anticipated with current gene therapy vectors and transduction conditions.

Description: Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to 200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

Description: Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3587 Poster Board III-524 The mechanism whereby neutrophils traffic from the circulation in response to G-CSF has remained unclear despite the observation of ourselves and others that there is a dramatic, yet transient, loss of circulating neutrophils shortly following the administration of G-CSF in humans, non-human primates, and mice (Gordon BC, et al. Exp Hematol. 35:872-8, 2007). To determine the role of the CD18 leukocyte integrin on neutrophils in the egress of neutrophils from the circulation, we used dogs with canine leukocyte adhesion deficiency (CLAD), a genetic disease in which a mutation in CD18 prevents CD18 surface expression. We selected CLAD dogs who had 5-10% CD18+ neutrophils following either matched littermate allogeneic transplant or autologous gene therapy for CLAD. Three CLAD dogs meeting these criteria were evaluated. Three carrier dogs served as controls. G-CSF was administered at 10μg/kg SQ to all six animals. Peripheral blood samples (EDTA) were taken immediately prior to G-CSF administration, and at 15, 30, 60, 120, 240 minutes, and 24 hours following G-CSF administration. Total white blood cell counts, neutrophil counts, and the number and percentage of CD18+ peripheral blood leukocytes were assessed. As anticipated, the control dogs had a 60% decrease in circulating neutrophils 30 minutes following G-CSF administration: the mean +/− standard of deviation (SD) absolute neutrophil baseline count decreased from 6806+/−1072/μL to 2727+/−767/μL. In five control animals the neutrophil nadir occurred at 30 minutes post-G-CSF, and in one control dogs it occurred 15 minutes following G-CSF administration. Experimental CLAD dogs had only a 35% decline in neutrophil numbers at 30 minutes, from a mean baseline of 6777+/− 672/μL to 4433+/−265/μL. In these dogs the neutrophil count returned to pre-G-CSF levels by 60 minutes post-G-CSF. By 24 hours after G-CSF, the neutrophil level was increased 3-fold from baseline. Immunophenotyping using an anti-CD18 and a canine specific anti-neutrophil PE conjugated antibody indicated that only the CD18+ neutrophils disappeared from the circulation following G-CSF administration. At baseline the transplanted CLAD dogs had a mean of 15.2+/−3.9% CD18+ peripheral blood leukocytes, of these 50.7+/−7.1% were CD18+ neutrophils. Thirty minutes following G-CSF administration the mean+/−SD percentage of CD18+ leukocytes declined to 13.7+/−3.7% with 33.4+/−5.8% being neutrophils. There was also a slight decline in CD14+CD18+ monocytes from 6.2 +/− 1.5% to 4.0 +/− 1.2%, which was not observed in the controls. There was no change in CD18- leukocyte numbers. The percentage of CD18+ neutrophils returned to baseline by 60 minutes and remained there at subsequent time points. These results demonstrate that the CD18 leukocyte integrin on circulating neutrophils mediates the transient neutropenia associated with G-CSF administration. Disclosures: No relevant conflicts of interest to declare.