An Observational Study from Long-Term AAV Re-administration in Two Hemophilia Dogs.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30-50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB.

Decreased hematopoietic progenitor cell mobilization in pearl mice.

Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin(-)), Sca(+) and c-kit(+) cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF.

Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Neutrophil elastase-processing defect in cyclic hematopoietic dogs.

OBJECTIVE: Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the beta3A subunit of adaptor protein complex-3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE. MATERIALS AND METHODS: Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85-98) and C-terminal (aa269-282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry. RESULTS: Antibody ELA85 (antibody to canine NE aa 85-98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269-282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active. CONCLUSION: Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Evaluation of clinical status, renal function, and hematopoietic variables after unilateral nephrectomy in canine kidney donors.

OBJECTIVE: To determine clinical status and renal and hematopoietic function after kidney donation and identify risks associated with kidney donation in dogs. DESIGN: Prospective study. ANIMALS: 14 dogs that underwent unilateral nephrectomy for kidney donation. PROCEDURES: Records were reviewed retrospectively to collect data regarding prenephrectomy clinicopathologic variables. Dogs were reexamined prospectively at various times after nephrectomy, and pre- and postnephrectomy CBC, serum biochemical analyses, urinalysis, and urine protein-to-urine creatinine ratio were compared. Six dogs had postnephrectomy renal volume determined ultrasonographically, and 4 of those dogs also underwent scintigraphic determination of glomerular filtration rate and renal biopsy. RESULTS: All dogs were clinically normal at the time of reevaluation. There were no significant differences between prenephrectomy and postnephrectomy values for BUN concentration or urine specific gravity. Mean postnephrectomy serum creatinine concentration was significantly greater than prenephrectomy concentration. Mean serum phosphorus concentration was significantly decreased after nephrectomy, and mean Hct, corpuscular volume, and corpuscular hemoglobin concentration were significantly increased after nephrectomy. Postnephrectomy renal volume was greatest in dogs < 12 months old at the time of surgery. Mean postnephrectomy glomerular filtration rate was 2.82 +/- 1.12 mL/kg/ min (1.28 +/- 0.51 mL/lb/min). Renal biopsy specimens obtained during and after nephrectomy were histologically normal. CONCLUSIONS AND CLINICAL RELEVANCE: Renal and hematopoietic variables were within reference ranges in dogs examined up to 2.5 years after unilateral nephrectomy. Compensatory renal hypertrophy was greatest in dogs < 1 year of age at donation. Donor age, along with histocompatability, may be an important factor in selecting dogs for kidney donation.

Hematopoietic chimerism induces renal and skin allograft tolerance in DLA-identical dogs.

OBJECTIVE: Hematopoietic chimerism, a state where donor and recipient bone marrow cells coexist, is associated with donor-specific tolerance. Nonmyeloablative bone marrow transplantation (BMT) has been shown to induce stable mixed hematopoietic chimerism in dog leukocyte antigen (DLA)-matched dogs. The potential for inducing renal and skin allograft tolerance with nonmyeloablative BMT was investigated in DLA-identical and DLA-haploidentical dogs in this study. MATERIALS AND METHODS: Renal allografts were performed in 8 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with (n = 8) and without (n = 4) simultaneous BMT. Skin allografts were performed in 2 DLA-identical and 4 DLA-haploidentical dogs after stopping CSP and MMF. Two DLA-identical control dogs received renal allografts without TBI, BMT, or immunosuppression with CSP and MMF. Molecular chimerism was determined with a PCR-based DNA microsatellite assay. Serum creatinine (Cr) concentration, urine specific gravity, and sequential renal biopsies were monitored to assess renal allograft function. RESULTS: Donor-type blood cells were first detected 4 weeks posttransplantation in both the myeloid and lymphoid lineages. Donor chimerism was present for at least 76 weeks in the DLA-identical dogs. Mixed chimerism was not observed in the DLA-haploidentical dogs or DLA-identical dogs that did not undergo BMT. The renal allografts were acutely rejected within 14 days in the 2 DLA-identical control dogs. There was long-term (> 5 yrs) renal allograft survival as evidenced by a normal (< 2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous BMT. Renal allograft inflammation was severe in the control dogs, mild to moderate in the DLA-haploidentical dogs, and minimal in the DLA-identical dogs. Donor-specific skin grafts were accepted in the DLA-identical dogs but rejected in the DLA-haploidentical dogs. Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal and skin allograft tolerance in DLA-identical and permit long-term renal allograft survival in DLA-haploidentical dogs. These findings suggest it may possible to obtain long-term allograft survival in DLA-identical and -haploidentical dogs without the need for chronic immunosuppressive therapy.

Renal allograft histopathology in dog leukocyte antigen mismatched dogs after renal transplantation.

OBJECTIVE: To evaluate allograft histopathology in dog leukocyte antigen (DLA)-mismatched dogs undergoing renal transplantation, with transient immunosuppression. STUDY DESIGN: Prospective study. ANIMALS: Ten healthy adult mongrel dogs. METHODS: Reciprocal renal transplantation and bilateral nephrectomy were performed. Immune conditioning consisted of nonmyeloablative (200 cGy), total body irradiation (TBI), bone marrow transplantation (BMT; 7 dogs), cyclosporine (CSA; 15 mg/kg every 12 hours), mycophenolate mofetil (MMF; 10 mg/kg every 12 hours) and intermittent prednisone (1 mg/kg every 12-24 hours). Biopsies were collected at transplantation, during full immunosuppression (44-90 days), and once medications were reduced or discontinued (228-580 days). Biopsies were evaluated for interstitial, tubular, vascular, and glomerular lesions. Blood urea nitrogen, creatinine, serum CSA concentrations, and clinical score were determined at each biopsy. RESULTS: Seven dogs survived >200 days (mean, 380 days). Transient CSA toxicity was suspected in 6 dogs. Lymphocytic, plasmacytic interstitial inflammation, and tubulitis progressed when immunosuppressive medications were decreased. All 7 dogs had histologic lesions consistent with some degree of allograft rejection at study end. CONCLUSION: Nonmyeloablative TBI, BMT, and short-term immunosuppression with CSA, MMF, and prednisone allowed renal allograft function and dog survival for >200 days. It appears unlikely that total drug withdrawal will be possible in unrelated DLA-mismatched dogs using this protocol. CLINICAL RELEVANCE: Transient immunosuppression with MMF, CSA, and prednisone along with BMT and nonmyeloablative TBI may make kidney transplantation a clinical reality for treatment of kidney failure in dogs. Initiating both MMF and CSA at lower dosages may potentially eliminate early renal allograft injury.

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

Correction of a large animal model of type I Glanzmann's thrombasthenia by nonmyeloablative bone marrow transplantation.

OBJECTIVE: The purpose of this study was to determine if nonmyeloablative bone marrow transplantation would induce stable hematopoietic chimerism that would correct the bleeding diathesis associated with type I Glanzmann's thrombasthenia (GT). METHODS: Three young dogs (less than 12 weeks of age) with GT were transplanted with DLA-matched bone marrow from littermates. Recipients received a sublethal dose (200 cGy) of total-body irradiation (TBI) prior to infusion with bone marrow (1-4 x 10(8) cells/kg). Recipient dogs were immunosuppressed with cyclosporine (15 mg/kg) and mycophenolate mofetil (10 mg/kg). Chimerism was determined by quantitation of donor microsatellite repeat polymorphisms in peripheral blood DNA and by flow cytometry to detect the presence of glycoproteins IIb and IIIa on platelets. Platelet function was assessed by a clot retraction test. RESULTS: One dog died one week posttransplant due to hemorrhage. Another dog died four weeks posttransplant from an unrecognized congenital heart defect and complications due to canine distemper virus infection. At the time of death, microsatellite analysis indicated 35 to 50% chimerism. Flow cytometry showed 20% of circulating platelets positive for glycoproteins IIb and IIIa. The third dog is alive and doing well approximately two years posttransplant. Hematopoietic chimerism has been sustained at 35 to 60% with approximately 30% of the platelets positive for glycoproteins IIb and IIIa. Platelet function is normal based on clot retraction. The animal does not have clinical signs of bleeding. CONCLUSIONS: These observations suggest that GT and perhaps other severe inherited platelet disorders can be corrected using nonmyeloablative bone marrow transplantation to establish partial chimerism with normal platelets in the platelet compartment.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) beta-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.