Mutations in the transcription factor GATA2 can cause MonoMAC syndrome, a GATA2 deficiency disease characterized by several findings, including disseminated nontuberculous mycobacterial infections, severe deficiencies of monocytes, natural killer cells, and B lymphocytes, and myelodysplastic syndrome. GATA2 mutations are found in similar to 90% of patients with a GATA2 deficiency phenotype and are largely missense mutations in the conserved second zinc-finger domain. Mutations in an intron 5 regulatory enhancer element are also well described in GATA2 deficiency. Here, we present a multigeneration kindred with the clinical features of GATA2 deficiency but lacking an apparent GATA2 mutation. Whole genome sequencing revealed a unique adenine-tothymine variant in the GATA2 -110 enhancer 116,855 bp upstream of the GATA2 ATG start site. The mutation creates a new E-box consensus in position with an existing GATA-box to generate a new hematopoietic regulatory composite element. The mutation segregates with the disease in several generations of the family. Cell type-specific allelic imbalance of GATA2 expression was observed in the bone marrow of a patient with higher expression from the mutant-linked allele. Allele-specific overexpression of GATA2 was observed in CRISPR/Cas9-modified HL-60 cells and in luciferase assays with the enhancer mutation. This study demonstrates overexpression of GATA2 resulting from a single nucleotide change in an upstream enhancer element in patients with MonoMAC syndrome. Patients in this study were enrolled in the National Institute of Allergy and Infectious Diseases clinical trial and the National Cancer Institute clinical trial (both trials were registered at www.clinicaltrials.gov as #NCT01905826 and #NCT01861106, respectively).

AbstractPatients with GATA2 deficiencyharbor de novo or inherited germline mutations in the GATA2 transcription factor gene, predisposing them to myeloid malignancies. There is considerable variation in disease progression, even among family members with the same mutation in GATA2. We investigated somatic mutations in 106 patients with GATA2 deficiency to identify acquired mutations that are associated with myeloid malignancies. Myelodysplastic syndrome (MDS) was the most common diagnosis (∼44%), followed by GATA2 bone marrow immunodeficiency disorder (G2BMID; ∼37%). Thirteen percent of the cohort had GATA2 mutations but displayed no disease manifestations. There were no correlations between age or sex with disease progression or survival. Cytogenetic analyses showed a high incidence of abnormalities (∼43%), notably trisomy 8 (∼23%) and monosomy 7 (∼12%), but the changes did not correlate with lower survival. Somatic mutations in ASXL1 and STAG2 were detected in ∼25% of patients, although the mutations were rarely concomitant. Mutations in DNMT3A were found in ∼10% of patients. These somatic mutations were found similarly in G2BMID and MDS, suggesting clonal hematopoiesis in early stages of disease, before the onset of MDS. ASXL1 mutations conferred a lower survival probability and were more prevalent in female patients. STAG2 mutations also conferred a lower survival probability, but did not show a statistically significant sex bias. There was a conspicuous absence of many commonly mutated genes associated with myeloid malignancies, including TET2, IDH1/2, and the splicing factor genes. Notably, somatic mutations in chromatin-related genes and cohesin genes characterized disease progression in GATA2 deficiency.

Trapped neutrophil syndrome is a rare congenital disease recognized in Border Collies and is characterized by persistent neutropenia with myeloid hyperplasia. The mechanism of neutropenia has not been described. We document the case of a young Border Collie diagnosed with trapped neutrophil syndrome based on clinical features, blood and bone marrow evaluation, and presence of the associated homozygous mutation. Results from flow cytometric and storage studies suggested lower neutrophil survival time. The dog had substantial neutrophilic inflammation in multiple organs, indicating that neutrophils could leave the marrow and enter tissues, making the term "trapped" neutrophil syndrome a misnomer.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the β2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

GATA2 deficiency was described in 2011, and shortly thereafter allogeneic hematopoietic stem cell transplantation (HSCT) was shown to reverse the hematologic disease phenotype. However, there remain major unanswered questions regarding the type of conditioning regimen, type of donors, and graft-versus-host disease (GVHD) prophylaxis. We report 59 patients with GATA2 mutations undergoing HSCT at National Institutes of Health between 2013 and 2020. Primary endpoints were engraftment, reverse of the clinical phenotype, secondary endpoints were overall survival (OS), event-free survival (EFS), and the incidence of acute and chronic GVHD. The OS and EFS at 4 years were 85·1% and 82·1% respectively. Ninety-six percent of surviving patients had reversal of the hematologic disease phenotype by one-year post-transplant. Incidence of grade III-IV aGVHD in matched related donor (MRD) and matched unrelated donor recipients (URD) patients receiving Tacrolimus/Methotrexate for GVHD prophylaxis was 32%. In contrast, in the MRD and URD who received post-transplant cyclophosphamide (PT/Cy), no patient developed grade III-IV aGVHD. Six percent of haploidentical related donor (HRD) recipients developed grade III-IV aGVHD. In summary, a busulfan-based HSCT regimen in GATA2 deficiency reverses the hematologic disease phenotype, and the use of PT/Cy reduced the risk of both aGVHD and cGVHD.

GATA2 deficiency is a bone marrow failure syndrome effectively treated with hematopoietic cell transplantation (HCT), which also addresses the predisposition to many infections (prominently mycobacterial). However, many GATA2-deficient persons who come to HCT also have prevalent and refractory human papilloma virus disease (HPVD), which can be a precursor to cancer. We analyzed 75 HCT recipients for the presence of HPVD to identify patient characteristics and transplantation results that influence HPVD outcomes. We assessed the impact of cellular recovery and iatrogenic post-transplantation immunosuppression, as per protocol (PP) or intensified/prolonged (IP) graft-versus-host disease (GVHD) prophylaxis or treatment, on the persistence or resolution of HPVD. Our experience with 75 HCT recipients showed a prevalence of 49% with anogenital HPVD, which was either a contributing or primary factor in the decision to proceed to HCT. Of 24 recipients with sufficient follow-up, 13 had resolution of HPVD, including 8 with IP and 5 with PP. Eleven recipients had persistent HPVD, including 5 with IP and 6 with PP immunosuppression. No plausible cellular recovery group (natural killer cells or T cells) showed a significant difference in HPV outcomes. One recipient died of metastatic squamous cell carcinoma, presumably of anogenital origin, at 33 months post-transplantation after prolonged immunosuppression for chronic GVHD. Individual cases demonstrate the need for continued aggressive monitoring, especially in the context of disease prevalent at transplantation or prior malignancy. HCT proved curative in many cases in which HPVD was refractory and recurrent prior to transplantation, supporting a recommendation that HPVD should be considered an indication rather than contraindication to HCT, but post-transplantation monitoring should be prolonged with a high level of vigilance for new or recurrent HPVD.

Background: We recently reported on the single-institution experience with allogeneic hematopoietic stem cell transplantation (HSCT) in 22 consecutive patients with GATA2 deficiency or the MonoMAC syndrome and observed a disease-free survival of nearly 90%. However, despite 10/10 HLA match of matched related donors (MRD) and matched unrelated donors (URD), there was a 25% incidence of grades III-IV acute graft-versus-host disease (GVHD) with tacrolimus/methotrexate (Tacro/MTX). In contrast, there was no grade III-IV acute GVHD in the haploidentical related donor (HRD) recipients, all of whom received GVHD prophylaxis with post-transplantation cyclophosphamide (PT/Cy) followed by tacrolimus/mycophenolate (tacro/MMF). Based on this initial experience, the protocol was subsequently amended to incorporate PT/Cy in all patients. We now report on this expanded cohort of a total of 59 patients, representing the largest experience with HSCT for GATA2 deficiency. Methods: This single-institution study was conducted at the National Institutes of Health Clinical Center between 2013 and 2020 (ClinicalTrials.gov Identifier: NCT01861106). Patients between 12 to 60 years of age were eligible if they had a deleterious mutation in the GATA2 gene, or clinical picture of the MonoMAC syndrome. Although the primary endpoints were engraftment and reversal of the clinical phenotype, a secondary endpoint was added to determine if PT/Cy in the MRD and URD reduced the incidence of grade III-IV aGVHD without compromising the overall and disease-free survival. Immune reconstitution at 100 days, 6, 12, 24, 36 months was analyzed. Results: 59 patients (median age at diagnosis 24 years; IQR 20-32) with GATA2 deficiency or the MonoMAC syndrome underwent allogeneic HSCT. MRD and URD recipients received busulfan for four days (targeted to an AUC 3600-4800) and fludarabine, whereas HRD recipients received two days of low dose cyclophosphamide, 5 days fludarabine, 200cGY total body irradiation, and two or three days of busulfan depending upon the presence or absence of clonal cytogenetic abnormalities. Donor sources included: 11 MRD, 31 URD, and 17 HRD. Median follow-up was 2 years [IQR 1-4], 88% (52/59) are currently alive. Seven deaths have occurred, including: persistent AML (n=1); infection (n=4); poor graft function and cardiac arrest (n=1) and from HPV-associated metastatic cancer (n=1). All of the patients who received HRD transplant are alive. Median time to neutrophil engraftment was 15 days (IQR 13-17) and 19.5 days (IQR 16-25) for platelets. Ninety three percent (55/59) of patients had blood test results after 100 days post-transplant to assess immune reconstitution; eighty one percent (45/55) had normal absolute monocyte counts, 67% (37/55) had normal absolute NK cell counts and 89% (49/55) had normal B cells counts. There was one case of primary graft failure in a recipient of an URD-transplant, and one secondary graft rejection in a recipient of HRD-transplant. Fifty-four percent (32/59) of patients had pre-HSCT myelodysplastic syndrome (MDS), and only two relapsed post-HSCT. Forty two percent (25/59) had abnormal cytogenetics pre-HSCT; amongst 23 patients with serial pre/post bone marrow cytogenetic evaluations, 19 had established normal cytogenetics post-HSCT. Forty five percent (19/42) of MRD/URD patients received Tacro/MTX for GVHD prophylaxis, and 31.5% (6/19) developed grade III-IV acute GVHD (aGVHD). In contrast, 55% (23/42) MRD/URD patients received PT/Cy for GVHD prophylaxis, and none developed grade III-IV aGVHD (p 0.0052). Five percent (1/17) of HRD recipients developed grade III-IV aGVHD. Forty two percent (8/19) of MRD/URD patients who received post-transplant Tacro/MTX had chronic GVHD, 4.3% (1/23) of MRD/URD patients who received PT/Cy developed cGVHD, and 5.8% (1/17) of HRD patients developed cGVHD within the first 2 years post-transplant. Conclusions: Allogeneic HSCT using a busulfan-based regimen in GATA2 deficiency results in nearly a 90% disease-free survival with long-term immune reconstitution. The use of PT-Cy reduced the risk of severe aGVHD and cGVHD and did not increase the risk of relapse or progression of MDS/AML. With earlier recognition of the disease, and the use of PT/Cy more broadly, these results are expected to continue to improve. Disclosures Notarangelo: NIAID, NIH: Research Funding.

BACKGROUND:Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice. OBJECTIVES:The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections. METHODS:Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin β2 subunit (ITGB2) gene. RESULTS:The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4. CONCLUSIONS:Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway.

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Compared to other classes of retroviral vectors used for HSPC gene therapy, foamy viral vectors (FVV) have distinct advantages, including their non-pathogenicity, a safer integration profile, and a high-efficiency transduction of quiescent cells. Proof-of principle of its therapeutic safety and efficacy in HSPCs was provided with the correction of canine Leukocyte Adhesion Deficiency (CLAD). Dogs with CLAD and individuals with LAD type 1 (LAD-1) suffer from recurrent and life-threatening infections caused by mutations in the β2 integrin CD18 subunit. In this study, G-CSF-mobilized CD34+ HSPCs were isolated from a 19 YO male with severe LAD-1 due to homozygous deletions in CD18. CD34+ cells were transduced for 16 hours at MOI of 0, 5, 10, and 20 with a FVV expressing a human codon optimized CD18 transgene. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 40-45% of cells. Thirty NSG mice were transplanted with CD18-FVV-transduced human LAD-1 HSPCs (~1.2 × 105 cells/mouse), and human cell engraftment was measured in murine BM 5 months after transplantation using flow cytometry. Human CD45+ cells were detected in all mice (average ~1%). Mice transplanted with mock-transduced (MOI=0) LAD-1 CD34+ cells showed a 3.4-fold, 2-fold and 1.4-fold lower engraftment compared to mice injected with CD34+ cells transduced with FVV at MOI 5 (p<0.01), 10 (p<0.05) and 20 (p>0.05), respectively, suggesting a selective homing/engraftment or survival/proliferative advantage of CD18+ cells. The inverse relationship between engraftment levels and MOI correlated with a gradual decrease in cell survival with increasing MOI, most likely due to toxicity from DMSO (required for FVV cryopreservation) during transduction; cell viabilities of 91%, 84%, 82% and 69% were obtained at MOI 0, 5, 10 and 20, respectively, indicating that further increase in MOI would lead to increasing toxicity. High-level, clinically relevant gene marking levels were obtained; the percentages of human cells expressing CD18 in the murine BM 5 months post-transplantation were 36.0 ± 3.9%, 33.9 ± 5.1%, and 44.5 ± 1.6% at MOI 5, 10 and 20, respectively. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in the majority of long-term repopulating HSPCs at all MOI tested. Flow cytometry-based lineage analysis of bone marrow from mice transplanted with FVV-transduced LAD-1 CD34+ cells revealed human CD18+ cells in all lineages, with a predominance in the CD13+ myeloid compartment (88.3 ± 4.5%) compared to other lineages, including CD20+ lymphoid (9.1 ± 3.9%), CD235a+ erythroid (0.2 ± 0.1%) and CD41+ megakaryocytic (0.6 ± 0.2%) lineages. Interestingly, human myeloid engraftment was superior in recipient mice engrafted with human CD18+ cells (81.5 ± 4.3%) compared to animals transplanted with non-transduced (CD18-) LAD-1 cells (65.3 ± 11.3%). Integration site analysis of engrafted human cells is ongoing. Thus, FVV-mediated transduction of human LAD-1 CD34+ cells leads to clinically significant levels of CD18 expression, supporting the use of this CD18-expressing FVV in a human clinical trial.

Strong viral enhancers in γ-retrovirus vectors (GV) have caused cellular proto-oncogene activation and leukemia in gene therapy trials, necessitating the use of cellular promoters in enhancer-less integrating vectors. However, data is now emerging that inadequate transgene expression from cellular promoters may limit successful correction. Vectors derived from foamy virus (FV), a nonpathogenic retrovirus, have a higher preference for non-genic integrations than GV/lentiviral vectors (LV), and may be less genotoxic. We constructed GV, LV and FV driven either by the spleen focus forming virus (SFFV) or the murine stem cell virus (MSCV) enhancer/promoters, all driving eGFP expression, and compared their relative genotoxicity using an in vitro immortalization assay on primary hematopoietic stem/progenitor cells (HSPC). In this assay, integration near a protooncogene/gene promoting cell proliferation results in quantifiable HSPC immortalization. Strong viral enhancer/promoters from SFFV or MSCV in FV caused a remarkably low immortalization of HSPC compared to analogous LV or GV: compared to the immortalization frequency of HSPC with the SFFV-GV in this assay, SFFV-LV and MSCV-LV had 12- and 14-fold lower immortalization frequency, while the SFFV-FV and MSCV-FV showed a 155- and 414-fold lower immortalization frequency, respectively. Immortalized clones had multiple (3-10) integrated copies. Integration site analysis of FV immortalized clones revealed a majority of integrants in non-gene regions; those in genic regions targeted cell proliferation or proto-oncogenes, as previously reported. FV has been previously reported to have 2-fold higher insertions in non-genic regions and higher, but nearly half the propensity to target promoters compared to GV. However, this remarkably reduced genotoxicity with FV was not explained by the integration pattern. We therefore hypothesized that FV backbone may contain sequences that have an enhancer blocking/insulator effect. Studies on chromatin insulators have shown that enhancer-blocking property of insulators is mediated via binding of CTCF to its consensus sequences within the insulator. Indeed, an in silico analysis for CCCTC-binding factor (CTCF) binding sites in the vector backbone sequences showed more predicted CTCF binding sites in the FV than in GV or LV (26, 8, and 6, respectively). To functionally validate the enhancer-blocking effect of the FV backbone and ensure that only effects of the vector backbone would be measured, without the confounding influence of integration site or the enhancer/promoter/transgene, we inserted SFFV-GV, SFFV-LV and SFFV-FV into a clinically relevant proto-oncogene, LMO2 , using CRISPR/Cas9, and assessed LMO2 expression. LMO2 upregulation has previously resulted in leukemias in the X-linked severe combined immune-deficiency and Wiscott-Aldrich syndrome (WAS) GV-mediated gene transfer trials; notably SFFV-GV was used in the WAS trial and caused leukemias in 8 of 10 patients from insertional oncogenesis. HeLa cells were transfected with the proviral donor plasmids and the guide-RNA/spCas9 plasmids and GFP+ cells sorted and cloned. Nearly all clones derived had one intact LMO2 allele, while the other alleles had GV/LV/FV proviral insertions. We next assessed LMO2 mRNA and protein expression in these clones. We found a minimal increase in LMO2 mRNA expression with SFFV-FV, in sharp contrast to significantly increased LMO2 expression with SFFV-GV and SFFV-LV by qRT-PCR ( Figure 1A ). Overall, the SFFV enhancer in GV demonstrated the greatest fold-increase in LMO2 expression (median increase of 280+/-23-fold over unmodified HeLa cells), followed by the SFFV enhancer in LV (median 200+/-27-fold increase). However, the same SFFV enhancer in FV only showed a 45+/-7-fold median increase in expression. Western blot analysis for LMO2 protein expression in three clones for each vector showed no detectable LMO2 expression in SFFV-FV clones, which was similar to baseline in mock (non-targeted) HeLa cells ( Figure 1B ). However, significantly higher LMO2 protein was detectable in GV and LV clones. Hence, the remarkably low genotoxic potential of FV, even those carrying strong viral enhancers appears to be, in large part, from an insulator property of FV sequences, making FV ideal for situations where high transgene expression, necessitating strong enhancers is required for a therapeutic effect. Disclosures No relevant conflicts of interest to declare.

In patients developing AML we could detect MDS1-EVI1 expression only after gene therapy with an increase over time up to the time point of AML development. As in immunodefi cient patients the diversity of T cell receptor (TCR) repertoire is usually reduced compared to a healthy situation and genetic translocations in leukemia patients frequently affect the TCR region, we further aimed to analyze the TCR repertoire of these patients before and after gene therapy, at the time of leukemia development and after chemotherapy. A new TCR sequencing approach established in our lab was used to determine the TCR gene usage of each patient, the presence of predominant TCR clones and the diversity of the TCR repertoire. So far, over 100 samples from blood and bone marrow of the patients were sequenced using the Illumina platform, retrieving more than 11 million sequences that are currently being analyzed.

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector.

Vector integration can lead to proto-oncogene activation and malignancies during hematopoietic stem cell gene therapy. We previously used foamy virus vectors to deliver the CD18 gene under the control of an internal murine stem cell virus promoter and successfully treated dogs with canine leukocyte adhesion deficiency. Here we have tracked the copy numbers of 11 specific proviruses found in these animals for 36-42 months after transplantation, including examples within or near proto-oncogenes, tumor suppressor genes, and genes unrelated to cancer. We found no evidence for clonal expansion of any of the clones, including those with proviruses in the MECOM gene (MDS1-EVI1 complex). These results suggest that although foamy virus vectors may integrate near proto-oncogenes, this does not necessarily lead to clonal expansion and malignancies. Additionally, we show that copy number estimates of these specific proviruses based on linker-mediated PCR results are different from those obtained by quantitative PCR, but can provide a qualitative assessment of provirus levels.

After administration of granulocyte colony-stimulating factor (G-CSF), there is a marked, albeit transient, drop in circulating neutrophils. To determine the role of leukocyte integrins in this disappearance, a dog having canine leukocyte adhesion deficiency (CLAD) or CLAD dogs who had undergone gene correction either by matched littermate allogeneic transplant or autologous gene therapy were evaluated. Shortly after G-CSF administration, a dramatic, yet transient, neutropenia was observed in the control littermates. This neutropenia was not as marked in the CLAD dogs. In all instances, it was CD18(+) neutrophils that preferentially egressed from the circulation. The association of CD18 with this rapid loss suggested leukocyte integrin activation after G-CSF administration. To determine the activation status of the integrin, a monoclonal antibody recognizing the activated α-subunit cation binding domain (mAb24) was used to evaluate human leukocytes after G-CSF administration. Mirroring the dramatic decrease in circulating neutrophil numbers, there was a dramatic and specific increase in the activation of the α-subunit after G-CSF expression on polymorphonuclear leukocytes. This activation, like the drop in neutrophil count, was transient. These results demonstrate that the leukocyte integrin on circulating neutrophils is transiently activated after G-CSF administration and mediates the transient neutropenia observed after G-CSF administration.

Abstract Abstract 2164 Following administration of granulocyte colony-stimulating factor (G-CSF) there is a marked but transient drop in circulating neutrophils. To determine the contribution of integrin activation to these events, a monoclonal antibody (mAb 24) that recognizes the activated α-subunit cation binding domain of the integrin family of receptors was used to evaluate human leukocytes following G-CSF administration. A single 480 mcg dose of human recombinant G-CSF (Neupogen, Amgen) was administered in the upper arm of six healthy human subjects (3 male, 3 female). EDTA and heparinized blood samples were collected prior to and at 15, 30, 60, 90, 120 and 240 minutes after injection. Mean absolute neutrophil count decreased from 2,557 ± 654/μl at baseline to 227 ± 193 (p<0.001) at 30 and 45 minutes. Flow cytometric analysis revealed a dramatic increase in binding of mAb24, which recognizes the occupied divalent cation binding site of the activated integrin α subunit, to neutrophils but not other circulating nucleated cells within 15 to 30 minutes after G-CSF administration, concurrent with a dramatic decrease in circulating neutrophil numbers. This activation, like the drop in neutrophil count, was transient, with a return to baseline status by 60 minutes. Intact parathyroid hormone (iPTH) levels were also evaluated. Within 30–90 minutes following G-CSF injection there was a significant drop in iPTH from a baseline of 52.5 ± 31.1 to a nadir value of 30.6 ± 20.9 pg/ml (p=0.01). A rebound in iPTH levels to 63.4 ± 27.0 pg/ml (p<0.001) occurred at 90 to 240 minutes. There were no significant changes in ionized calcium or magnesium levels. These results demonstrate that the integrin on circulating neutrophils is transiently activated following G-CSF administration and that this activation may mediate the transient neutropenia seen in this setting. In addition, alterations in PTH levels occur following G-CSF administration. As it is known that iPTH influences the hematopoietic stem cell (HSC) niche, fluctuations in iPTH levels following G-CSF administration may play a role in HSC expansion. Disclosures: No relevant conflicts of interest to declare.

Proto-oncogene activation caused by retroviral vector integration can cause malignancies in gene therapy trials. This has led investigators to search for less genotoxic vectors with minimal enhancer activity and a decreased risk of influencing neighboring chromosomal gene expression after integration. We previously showed that foamy virus (FV) vectors expressing the canine CD18 gene from an internal murine stem cell virus (MSCV) promoter could cure canine leukocyte adhesion deficiency (LAD). Here, we have repeated these studies using a FV vector expressing canine CD18 from a phosphoglycerate kinase (PGK) gene promoter. In vitro analysis showed that this vector did not contain an enhancer that activated neighboring genes, and it expressed CD18 efficiently in canine neutrophils and CD34+ cells. However, dogs that received hematopoietic stem cells transduced with the PGK-CD18 vector continued to suffer from LAD, and sometimes died prematurely of the disease. These studies show that the PGK promoter cannot effectively replace the MSCV promoter in CD18-expressing FV vectors, and they suggest that vectors containing a strong promoter–enhancer may be necessary for the treatment of human LAD.

Abstract Abstract 3779 Leukocyte adhesion deficiency (LAD) caused by CD18 deficiency is an autosomal recessive immunodeficiency which has been described in humans, Irish Setter dogs, Holstein cattle, and transgenic mice. We report here on the characterization of the clinical features and leukocyte function abnormalities of a novel feline model of leukocyte adhesion deficiency. The proband was a 5 year old male domestic longhair cat with a life-long history of infections, inflammation and granulomas. Severe gingivitis was recognized at 8 weeks of age which progressed to peridontitis and loss of all deciduous and adult teeth before 1 year of age. Over the next years this cat experienced recurrent severe upper respiratory infections, gastroenteritis, anterior uveitis, various abscesses, and epidermal sloughing with sepsis secondary to small grooming lacerations. The various illnesses seemed to be responsive to various long-term antibiotic as well as glucocorticoid treatments. Complete blood cell counts revealed a persistent severe leukocytosis (50-91,000/μl) due to mature neutrophilia with few toxic changes, and mild lymphocytosis, monocytosis and eosinophilia. Serum immunoglobulin concentrations were normal. Compared to feline control cells, neutrophils of the proband did not express any CD18 on the cell surface as determined with two monoclonal anti-CD18 antibodies but normal CD45 expression. Adhesion of affected neutrophils with and without PMA activation was severally impaired; the adhesion of normal feline neutrophils could be completely blocked by the anti-CD18 antibody. In a proliferation assay the proband's T-cells responded weakly at 1 pg SEA but near normally at 100 pg SEA, suggesting a CD18-independent T-cell response in cats. Sequencing of the feline CD18 β-integrin gene is being completed. In conclusion, this naturally-occurring feline model of CD18 deficiency exhibits similar features to LAD in other species. However, feline LAD seems clinically milder allowing for a longer life expectancy possibly due to T-cell independent activities. This LAD model may be helpful in developing and assessing novel therapies. Disclosures: No relevant conflicts of interest to declare.