ALBERT

Description: Background : Caspofungin (CAS) is a novel echinocandin, which is approved for several antifungal indications in adults. Although the compound is not licensed in pediatric patients (pts), it is being used in children with refractory infections or intolerance to standard antifungal agents. Methods: We conducted a multicenter retrospective survey to obtain data on clinical use, safety, and outcome in immunocompromised pediatric pts who received therapy with CAS. Results : The survey identified 71 immunocompromised children and adolescents [mean age (range) 11.4 years (5 months –26 years)]. Out of the 29 females and 42 males, 53 suffered from hematological malignancies, 10 from bone marrow failure syndromes, 3 from solid tumors, 2 from congenital immunodeficiency, and 3 from non-malignant hematological disorders. Forty-two pts (59%) had undergone allogeneic blood stem cell transplantation, and 36 pts (51%) had an ANC of less than 500/μL at baseline. CAS was administered for proven (n=18), probable (15), and possible (20) invasive fungal infection, or as empirical antifungal treatment (18). All but one pt had received prior systemic antifungal therapy with amphotericin B (52) and/or triazoles (41). The 71 pts received CAS for a mean duration of 43.5 days (range, 2–218) as single agent (22) or in combination with other antifungal agents (49). The mean maintenance dosage of CAS was 1.2 mg/kg (range, 0.4–2.9) or 34.8 mg/msqu (range, 16.3–57.5). In none of the pts, CAS was discontinued prematurely due to clinical or laboratory adverse events. Clinical adverse events were described in 35 children (49%), mostly fever (28), nausea and vomiting (18), diarrhea (8), and headache (5). Increases in hepatic or renal function parameters were frequent in these pts that received multiple other therapeutic compounds. Whereas at the end of treatment (EOT), mean GPT and GOT were slightly elevated (from 41 at baseline to 61 U/L at EOT; p=0.002 and from 29 to 80 U/L, p=0.001, respectively), mean serum creatinine, bilirubin, and alkaline phosphatase values were not different from baseline. Complete responses, partial responses, or stabilization were observed in 4/8/3 of 18 evaluable pts with proven, in 4/2/3 of 13 pts with probable, and in 3/8/2 of 15 pts with possible invasive fungal infection. Of 16 evaluable pts who received CAS empirically therapy, 10 successfully completed therapy. Overall survival was 74% at EOT and 67% at three months post EOT (65 and 60 evaluable pts, respectively). Conclusions: The data of this retrospective survey show that CAS displays acceptable safety and tolerance and may have useful antifungal efficacy for second line treatment of severely immunocompromised pediatric patients.

Description: Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical γ-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and γ-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

Description: About 10–15% of acute myeloid leukemia (AML) arise secondary after chemotherapeutic treatment and/or radiation for a primary malignant disease and are therefore called therapy-related AML (t-AML). Overall, t-AMLs respond less well to treatment than their de novo counterparts. It has also been shown that the karyotype of the leukemic blasts is an independent prognostic parameter in t-AML. Here we assessed the gene expression profiles of 53 t-AML cases compared to a matched patient cohort of 53 de novo AML patients using Affymetrix DNA-oligonucleotide microarrays (HG-U133 chip design). The following cohort was analyzed (de novo and t-AML each): 2 t(15;17), 3 inv(16), 4 t(8;21), 4 normal karyotypes, 5 other intermediate karyotypes, 2 t(8;16), 5 inv(3), 20 t(11q23)/MLL, and 8 complex aberrant karyotypes. First, we aimed at mining for specific patterns separating de novo from t-AML. Both unsupervised, i.e. hierarchical clustering and principal component analysis, and supervised data analysis algorithms could not identify a signature that robustly separated de novo from therapy-related cases. Then the algorithm was changed to analyze both patient cohorts stratified according to favorable cytogenetics, i.e. t(15;17), t(8;21), inv(16), intermediate cytogenetics, i.e. inv(3), and unfavorable cytogenetics, i.e. t(11q23)/MLL and complex aberrant karyotypes. This resulted in gene expression signatures where de novo and t-AML patients were closely related to each other but separated within genetic subgroups. Particularly in t(11q23)/MLL, complex aberrant karyotypes and t(8;21) samples, de novo cases intercalated with therapy-related cases. When a supervised classification algorithm (Support Vector Machine) was trained to predict the subtype for both de novo and t-AML samples misclassifications were observed. However, these misclassifications mainly occurred within cytogenetic subgroups, e.g. therapy-related AML with complex aberrant karyotype was classified as de novo AML with complex aberrant karyotype, or de novo t(11q23)/MLL as therapy-related t(11q23)/MLL and vice versa. In contrast, an analysis only focusing on cytogenetic subtypes and grouping de novo and t-AML cases together clearly identified signatures related to the biologically distinct AML subtypes. Taken together, we were not able to define a specific expression profile globally associated with therapy-related AML. Instead, t-AML cases of distinct cytogenetic subgroups were demonstrated to be similar to their de novo counterpart. Despite known differences in the genetic events leading to the malignant transformation, i.e. leukemogenic exposures in t-AML through alkylating agents, topoisomerase II inhibitors, or radiation both de novo and t-AML can be considered as biologically identical diseases. Thus, given the underlying biology in AML one should separate patients in clinical trials according to cytogenetics and their history as t-AML or de novo as suggested by the WHO classification. However, differences in prognosis may not be explainable by the respective gene expression profiles in both groups.

Description: Bone marrow (BM) contains hematopoietic stem cells (HSCs), which differentiate into every type of mature blood cell; endothelial cell progenitors; and marrow stromal cells, also called mesenchymal stem cells (MSCs), which can differentiate into mature cells of multiple mesenchymal tissues including fat, bone, and cartilage. Recent findings indicate that adult BM also contains cells that can differentiate into additional mature, nonhematopoietic cells of multiple tissues including epithelial cells of the liver, kidney, lung, skin, gastrointestinal (GI) tract, and myocytes of heart and skeletal muscle. Experimental results obtained in vitro and in vivo are the subject of this review. The emphasis is on how these experiments were performed and under what conditions differentiation from bone marrow to epithelial and neural cells occurs. Questions arise regarding whether tissue injury is necessary for this differentiation and the mechanisms by which it occurs. We also consider which bone marrow subpopulations are capable of this differentiation. Only after we have a better understanding of the mechanisms involved and of the cells required for this differentiation will we be able to fully harness adult stem cell plasticity for clinical purposes. (Blood. 2003; 102:3483-3493)

Description: Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose–dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 × 1011 vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 × 1011vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the -skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol.

Description: Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.

Description: Based on studies in mice, hemophilic dogs, and non-human primates demonstrating long-term (〉5 yrs) expression of Factor IX (FIX) after infusion of an AAV vector expressing FIX into the portal vein or the hepatic artery, we undertook a Phase I dose escalation study of AAV-FIX in humans with severe hemophilia B. The first two doses, 2x1011 vg/kg, and 1x1012 vg/kg, were safe but subtherapeutic. Two subjects treated at a dose of 5x1012 vg/kg showed detectable circulating levels of FIX (up to 11.8% and 3% respectively), but expression was transient and accompanied in one case (subject E) by a transient asymptomatic transaminitis. There was never evidence of a FIX inhibitor. Two differences between the large animal models and humans with the disease were hypothesized to contribute to the difference in duration of expression; long-term in hemophilic dogs, short-term in hemophilic humans. First was pre-existing immunity to wild-type AAV-2, which infects humans, but not dogs; and the other was prior exposure to viral hepatitis , found in humans but not in animals. To further assess the roles of viral hepatitis and of the immune response to AAV-2, we treated an additional subject (subject G) at a dose of 1x1012 vg/kg. This subject was 20 yrs. of age and had never been infected with hepatitis. Nevertheless, his transaminases began to rise 3 weeks after vector injection, peaked 6 weeks after injection, and resolved spontaneously as had been seen in subject E. In subject G, magnitude of the peak ALT response was 5-fold less than that found in subject E (5-fold higher dose). Both subjects had similar and low baseline anti-AAV antibody titers. Immune response to AAV-2 was assessed by ELISpot at serial time points before and after vector injection in subject G. The subject’s PBMCs were incubated with a peptide library arrayed in a matrix of 24 pools, each containing 12 peptides of 15-mers overlapping by 10 and spanning the entire VP-1 protein. There was no detectable IFN- γ secretion in response to AAV-2 peptides at baseline, although there was a strong IFN- γ response to PHA. Two weeks after vector infusion, three pools elicited IFN- γ secretion from the subject’s PBMCs. Response to the same pools of peptides, but not to other pools, was repeatedly detected over the next 6 weeks. By week 12, IFN- γ responses were no longer detectable. The matrix array allowed identification of two specific AAV-2 capsid peptides as the T cell immunoreactive epitopes. These peptides are highly conserved in AAV serotypes 1–8. Similar experiments were conducted with a FIX peptide library and demonstrated no response. These data are consistent with a model in which a T cell response to AAV capsid epitopes results in elimination of the transduced cells. This response is only briefly detectable in PBMCs, and hepatitis is not an important risk factor. These immune responses may limit use of standard serotypes of AAV for gene transfer into human liver. Transient immunomodulation may prevent these responses.

Description: The use of gene replacement therapy is an attractive approach for the treatment of the genetic bleeding disorder hemophilia B (caused by mutations in the coagulation factor IX, FIX, gene). A major concern with this type of procedure is the potential for a host immune response to the therapeutic gene product, which would render treatment ineffective. Previously, we observed inflammatory, cytotoxic T lymphocyte, and antibody responses to a human FIX (hFIX) transgene product after intramuscular (IM) delivery via an E1/E3-deleted adenoviral vector (Ad-hFIX) in C57BL/6 mice. Different from this Th1-biased immune response, IM injection of adeno-associated viral (AAV) vector, a Th2-biased, non-inflammatory response led to antibody-mediated neutralization of hFIX expression, without CTL activation. In contrast to these observations on muscle-directed vector administration, hepatic AAV-hFIX gene transfer induced immune tolerance to the transgene product (JCI 111:1347). Lack of anti-hFIX formation was demonstrated even after challenge with hFIX in adjuvant. In order to examine the effect of tolerance induction on CD8+ T cell-mediated cellular immune responses, we performed the following experiments. C57BL/6 mice (n=4 per experimental group) received IM injections of AAV-hFIX vector (serotype 1) in one hind limb and/or Ad-hFIX vector in the contra-lateral leg. In the latter case, inflammation (as determined by H&E histological evaluation), CD8+ T cell infiltrate and destruction of hFIX expressing muscle fibers were obvious in both legs because of the Ad-hFIX mediated activation of CTL to hFIX. CD8+ T cell responses were strongest in Ad-hFIX transduced muscle at day 14 and in the AAV-hFIX leg at day 30. Expression of hFIX as determined by immunohistochemistry became undetectable in Ad-hFIX injected muscle by day 30, but was not completely eliminated in AAV-hFIX transduced muscle. Injection of AAV-hFIX only, did not cause inflammation of muscle tissue or CD8+ cell infiltrate. When the identical experiment was carried out in C57BL/6 mice that were expressing hFIX from hepatic gene transfer via the AAV serotype 2 vector (performed 6 weeks earlier), a substantial increase in systemic hFIX expression was observed after IM administration of the Ad and AAV-1 vectors (again injected into contra-lateral legs). However, a portion of the increased expression was subsequently lost, which correlated with inflammation and CD8+ T cell infiltrate of the Ad-hFIX transduced muscle. Interestingly, no (3/4 mice) or only minor (1/4 mice) infiltrate was observed in AAV-hFIX injected muscles. Consequently, hFIX expression persisted in the AAV, but not the Ad transduced legs. Presumably, CTL responses to adenoviral antigens were sufficient to target Ad-hFIX transduced muscle despite tolerance to the transgene product. In contrast to control mice, hepatic tolerized animals failed to form anti-hFIX after challenge by IM injection of these viral vectors. Moreover, inflammatory and destructive cellular immune responses to the transgene product were successfully prevented by hepatic tolerance induction, indicating that tolerance induced by gene transfer to the liver affects cellular as well as antibody-mediated responses and extents to tissues other than liver.

Description: The prolonged presence of unusually large VWF multimers due to reduced or absent proteolytic degradation by ADAMTS13 has been recognized as the main cause for thrombotic thrombocytopenic purpura (TTP). Congenital TTP is associated with gene defects whereas acquired TTP, is caused by autoantibodies to ADAMTS13. Such antibodies might block ADAMTS13 enzymatic activity or enhance its clearance from the circulation. There is an unknown correlation between ADAMTS13 activity and ADAMTS13 antigen level. Here we report for the first time the development of an ELISA to quantify ADAMTS13 antigen levels in human plasma derived from healthy controls, and patients diagnosed with thrombotic microangiopathies (TMAs) like TTP or the haemolytic uraemic syndrome (HUS). To set up the ELISA system, we used an affinity purified polyclonal rabbit anti-human ADAMTS13 IgG fraction to capture the human plasmatic ADAMTS13 and the same antibody preparation, although horseradish-peroxidase-labeled, as detection antibody. Quantification of bound ADAMTS13 was performed using ADAMTS13 depleted human plasma as matrix and recombinant human ADAMTS13 as standard. An interference of ADAMTS13 autoantibodies with the ELISA system was excluded by spiking experiments. In total we analyzed 94 individuals, 25 healthy donors and 69 patients with the clinical diagnosis of TMA. In the TMA group 17 patients were suffering from hereditary TTP/HUS, 40 patients had acquired TTP and 12 patients had HUS. In healthy donors the ADAMTS13 activity range was 0.46-1.25U/ml and the normal range obtained for ADAMTS13 antigen levels was 680–1350ng/ml (mean 1010ng/ml). Patients with diagnosis of hereditary TTP/HUS had low or undetectable ADAMTS13 activities and antigen levels