Introduction: Post transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. Treatment has proved difficult and includes dose reduction of immune suppression, combination chemotherapy, surgical excision and radiotherapy. Recently the monoclonal antibody Rituximab, has been added to the therapeutic choice, but how and when to use it is not clear. Previous studies have reported limited success with Rituximab alone plus decreased immune suppression (Choquet et al Blood2003:102:11, 986). We retrospectively analysed 40 patients who developed PTLD post-renal transplant. Methods Patients were evaluated who received a renal transplant between 1981–2000. 30 patients were male and 10 female (median Age 46 years, range 21–70). The median time to development of PTLD was 54 months (range 1m–192m). Diagnostic samples were analysed by immunohistochemistry and clonality assessed by molecular analysis. For patients with monomorphic High grade histology an 18 FDG-PET scan was performed at diagnosis and an interim scan after 2 cycles of therapy (CHOP or CHOP/R, 21 day cycle). Treatment groups received a) no treatment due to rapidly progressive disseminated disease, b) decreased immune suppression alone, c) decreased immune suppression plus combination chemotherapy, d) decreased immune suppression plus single agent rituximab, e) decreased immune suppression plus combination chemo-immunotherapy (CHOP/R, all late B-PTLD > 3 years), or f) surgery (n=1). Results Median Overall survival for all patients was 15 months (range 0.5–96 months). For different patient groups according to treatment, median OS was as follows: For no treatment group (n=5, OS 2.5 months), for decreased immune suppression group (n=8, OS 53.5 months, all less aggressive polymorphic histology), for decreased immune suppression plus chemotherapy (n=16) median OS was 14 months, for the decreased immune suppression group plus single agent Rituximab (n=4) median OS 22months, for the decreased immune suppression group plus chemo immunotherapy (n=6) median OS is 34.5months (range 8–48months) with 6/6 (100%) patients alive and well in CR. No patients treated with CHOP/R suffered any adverse side effects or renal co-morbidity. 10 patients underwent an initial diagnostic PET scan and an interim PET scan .9/10 patients interim PET was negative and 1 patient obtained a PR which become negative at the end of treatment. All patients with a negative interim PET currently remain in CR (100%) at a median follow up of 30 months. Conclusions The treatment of B-PTLD is difficult. Our series of patients now includes long term follow up on patients who either received Rituximab alone plus decreased immune suppression or chemo-immunotherapy plus decreased immune suppression. With a median follow up of 34.5 months, all CHOP/R treated patients are in CR. These results are encouraging and appear to offer significant improvement over previous treatments modalities i.e. chemotherapy only. For PTLD with high-grade histology a negative interim PET scan was highly predictive of prolonged OS and CR status.

Introduction: Thalidomide analogues such as CC-4047 (Actimid, Celgene) have shown efficacy in treatment of relapsed myeloma but the exact mode of action remains unclear (Streetly M, et al ASH 2003). Preliminary work (Schey S, JCO 2004) suggests that thalidomide analogues can activate T-cells. We have demonstrated that measurement of serum free light chains at day 7 of treatment with Actimid can indicate likelihood of response (Patten P, et al, ASH 2003). Interleukin-2 (IL-2) secretion is increased in patients with myeloma on Actimid although this is not associated with clinical response (Ahsan G, et al Haematologica. Suppl.1 2005). We have now looked at expression of activation markers on T cells following 28 days of CC-4047. Aim: The aim of this study was to see if CC-4047 induces activation of T-cells and whether this correlated with clinical response as assessed by use of the serum free light chain (SFL) assay. Patients and Method: 17 Patients (median age 59, range 34–75 years) participated in a phase I/II dose escalation study with CC-4047. All patients received treatment with oral CC-4047, on alternate day schedules. Serum was collected for IL-2 and serum free light chain. EDTA samples were taken for FACS analysis, which was performed on separated lymphocytes. 4 colour flow cytometry was performed using CD3/CD4/CD8/CD56 and CD69 monoclonal antibodies (Beckman Coulter, UK). Analysis for IL-2 and serum free light chains were performed as previously described (Ahsan et al Haematologica 2005 suppl.1) Results: Using conventional assessment 9/17 patients had evidence of response (CR, VGPR, PR) and 8 had stable disease ( Conclusions: This study showed that CD69 expression is increased on T cells in myeloma patients treated with CC-4047. This was detectable at day 28 of treatment. There did not appear to be a correlation between CD69 expression at day 28 and change in serum free light chain ratio. In line with other studies we have identified T cell activation in patients treated with the thalidomide analogue CC-4047. This does not appear to correlate with likelihood of response and it may be that response is due to activation of NK cells or that sustained activation of T-cells after 28 days is important for response. Further work is on-going to address these questions.

Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F. IX missense mutation (Herzog et al., Nat. Med. 1999; 5: 56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF. IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF. IX responses occurred at vector doses of 2x10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

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 gamma-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 gamma-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.

The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functional coagulation factor IX (hemophilia B) by intramuscular (IM) administration of an adeno-associated viral (AAV) vector. A potentially serious complication of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setting of null mutations in the factor IX gene (F9). Here, we describe hemophilia B mice with a large F9 deletion that form inhibitors within 1 to 2 months after IM administration of an AAV vector expressing mouse F9 or after repeated intravenous infusion of mouse F9 concentrate. In both cases, inhibitors are primarily IgG1 immunoglobulins representing a Th2-driven humoral immune response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in complete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with the immunosuppressive drug cyclophosphamide. These data have direct relevance for design of clinical trials and strategies aimed at avoiding immune responses against a secreted transgene product.

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.

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4+ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.

HaemophiliaVolume 4, Issue 5 p. 699-703 Gene therapy for haemophilia: how far have we come? P. A. Fields, P. A. Fields Katherine Dormandy Haemophilia Centre, Department of Haematology, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, UKSearch for more papers by this authorK. J. Pasi, K. J. Pasi Katherine Dormandy Haemophilia Centre, Department of Haematology, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, UKSearch for more papers by this author P. A. Fields, P. A. Fields Katherine Dormandy Haemophilia Centre, Department of Haematology, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, UKSearch for more papers by this authorK. J. Pasi, K. J. Pasi Katherine Dormandy Haemophilia Centre, Department of Haematology, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG, UKSearch for more papers by this author First published: 12 December 2003 https://doi.org/10.1046/j.1365-2516.1998.00175.xCitations: 1 K. J. Pasi Tel: 0171 794 0500 x4140; fax: 0171 830 2178; email: [email protected] Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Peake I. The role of gene therapy in haemophilia. Haemophilia 1995; 1: Suppl. 1, 40–3. 2 Dai Y, Roman M, Naviaux RK, Verma IM. Gene therapy via primary myoblasts:longterm expression of factor IX protein following transplantation in vivo. Proc Nat Acad Sci USA 1992; 89: 10 892–5. 3 Yao SN, Kurachi K. Expression of human factor IX in mice after injection of genetically modified myoblasts. Proc Nat Acad Sci USA 1992; 89: 3357–61. 4 Verma IM, Dai Y. Progress in gene therapy. Proc XXI Int Congr World Federation of Haemophilia 1994; 15. 5 Kay MA, Rothenburg S, Landen CN, et al. In vivo hepatic gene therapy of Haemophilia B: sustained partial correction in factor IX deficient dogs. Science 1993; 262: 117–9. 6 Kay MA, Landen CN, Rothenburg SR, et al. In vivo hepatic gene therapy: complete albeit partial correction of factor IX deficiency in haemophilia B dogs. Proc Nat Acad Sci USA 1994; 91: 2353–7. 7 Yang Y, Nunes FA, Berencsi K, Gonczol E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenovirus improves the prospect for gene therapy in cystic fibrosis. Nature Genetics 1994; 7: 362–9. 8 Herzog R, Hagstrom J, Kung S, et al. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Nat Acad Sci USA 1997; 94: 5804–9. 9 Gu H, Marth J, Orban P, Mossmann H, Rajewsky K. Deletion of a DNA polymerase gene segment in T cells using cell type specific gene targeting. Science 1994; 265: 103–6. Citing Literature Volume4, Issue5September 1998Pages 699-703 ReferencesRelatedInformation