ALBERT

Beschreibung: Background : Recipients of hematopoietic stem cell transplantation (HSCT) can be severely immunocompromised, predisposing to opportunistic infections including cytomegalovirus (CMV). While adoptive transfer of ex vivo expanded donor antiviral T-cells can prevent viral disease, this approach is not frequently used clinically because cell culture and selection procedures are lengthy, labor-intensive, and expensive. We describe an alternative approach. A single inoculation of a strongly immunogenic, but safe, live-attenuated, Listeria-based vaccine (Lm-MCMV) can rapidly drive extensive in vivo expansion of anti-murine CMV (MCMV) CD8+ T-cells during immune reconstitution following HSCT. Methods : The Lm-MCMV vaccine is derived from a genetically defined Listeria monocytogenes ΔactAΔinlB vaccine strain (Brockstedt et al. PNAS in press) that is attenuated by 4-logs in a mouse virulence assay, as compared to wild-type Listeria. Lm ΔactAΔinlB was engineered to express and secrete the MCMV H-2b immunodominant peptide HGIRNASFI within an ovalbumin scaffold. C57BL/6 (H-2b; CD45.2) HSCT recipients were conditioned with 11 Gy irradiation on day -1, and injected with 5x106 T cell depleted (TCD) C57BL/6 bone marrow cells on day 0. Selected mice simultaneously received 3x107 splenocytes from PepBoy (H-2b; CD45.1) donors that were either naive or immunized with 1x107 colony forming units (cfu) Lm-MCMV on day -7. On day +21, selected groups were vaccinated with 1x107 cfu Lm-MCMV, and sacrificed up to 14 days later. MCMV-specific CD8+ T-cells were quantified by intracellular cytokine and tetramer staining. Residual viable Lm-MCMV vaccine in the spleen, liver, and brain of BMT recipients was measured by plating on BHI agar media. Results : No mortality occurred following vaccination. Viable Lm-MCMV, which peaked the day after immunization in the liver (avg = 6.1x104 cfu) and spleen (avg = 1.4x105 cfu) of HSCT recipients, were completely cleared within 5 days of inoculation. Viable Listeria were not isolated from brain. Without vaccination at day +21, none of the HSCT recipients had detectable anti-MCMV CD8+ T cells. In contrast, immunization with Lm-MCMV at day +21 lead to marked antiviral T-cell expansion in all groups. HGIRNASFI-specific CD8+ T-cells averaged 2.4% of total CD8+ T-cells in vaccinated TCD marrow recipients. Levels were significantly higher in recipients of TCD marrow plus naive (7.1%) or immune (19.0%) donor splenocytes. Conclusions : Immunization of BMT mice with live-attenuated Lm-MCMV was safe. All HSCT recipients survived immunization and rapidly cleared viable Listeria. A single Lm-MCMV immunization during the period of immune reconstitution following HSCT was also effective, driving extensive antiviral T-cell expansion. In HSCT recipients of TCD marrow alone, vaccination produced levels of HGIRNASFI-specific CD8+ T-cells (2.4%) comparable to that seen with adoptive immunotherapy. Co-transplantation of naive or immune donor splenocytes lead to significantly higher levels of antiviral CD8+ T-cells following vaccination (7.1% and 19%, respectively). This approach could represent a broadly applicable alternative to adoptive immunotherapy. Future studies will focus on optimizing vaccination strategies, including use in allogeneic transplantation.

Beschreibung: Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for 75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (〉108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (〉16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

Beschreibung: Background: We previously described a live-attenuated (L/A) Listeria monocytogenes (Lm)-based vaccine encoding murine CMV (MCMV) epitopes (Lm-MCMV) that effectively drives expansion of antiviral CD8+ T-cells in wild-type mice and following bone marrow transplantation (BMT). We now show similar efficacy with increased safety using a non-replicating killed but metabolically active Lm vaccine (KBMA-Lm-MCMV). Furthermore, we demonstrate that resulting antiviral T-cells persist long-term (〉200 days) and exert functional antiviral activity. Methods: The L/A Lm-MCMV vaccine is derived from a genetically defined Lm ΔactA/ΔinlB vaccine strain (Brockstedt et al., PNAS101:13832, 2004) that is attenuated by 4-logs in a mouse virulence assay. The non-replicating KBMA vaccine is derived from ΔactA/ΔuvrAB bacterial mutants treated with amotosalen, a proprietary DNA-crosslinking psoralen. Both vaccines express the MCMV H-2b immunodominant peptide HGIRNASFI. C57BL/6 (H-2b) BMT recipients were conditioned with 11 Gy irradiation on day -1, and injected with 5x106 T cell depleted (TCD) C57BL/6 bone marrow cells on day 0. Selected mice received 3x107 splenocytes from syngeneic donors that were immunized 7 days previously with 107 colony forming units (cfu) Lm-MCMV. BMT mice were vaccinated with L/A Lm-MCMV on day 21 after transplant, or with KBMA-Lm-MCMV on days 1, 2, and 3 after transplant. Results: Vaccination of wild-type C57BL/6 mice with 107 cfu L/A Lm-MCMV (〈 0.03 LD50) induced anti-HGIRNASFI CD8+ T-cells to a peak of 9.7% (+/- 1.6%) of total CD8+ T-cells 7 days later. In BMT mice, L/A Lm-MCMV vaccination was delayed to day 21 post-transplant to reduce mortality, and lead to peak CTL responses of 17.6% of total CD8 within 7 days. Interestingly, the novel non-replicating KBMA vaccine could be administered immediately after BMT without mortality, but nonetheless antiviral CTLs still expanded to 16% of total CD8 by 7 days after BMT. At 〉 200 days after vaccination of wild-type or BMT mice, HGIRNASFI-specific CD8+ T-cells still accounted for 0.5 - 5% of all CD8+ T-cells. Interestingly, mice convalescent after a similarly remote MCMV infection (106 pfu) had significantly lower levels of antiviral T-cells (0.1-1%; p 〈 0.05). Following either Lm-MCMV vaccination or MCMV infection the majority of tetramer-positive cells were CD44hi and CD62Lhi, consistent with the central memory subset of CD8+ T-cells. Using In vivo CTL assays, Lm-MCMV vaccination produced specific anti-HGIRNASFI lytic activity averaging 96.7% (+/− 4.0%) at 〉 200 days after vaccination, which was similar to that seen after MCMV infection. Vaccination also significantly reduced viral loads by 46% (p 〈 0.026) following experimental MCMV infection. Conclusions: Vaccination with the L/A or KBMA Lm-MCMV vaccine rapidly produced high levels of anti-MCMV CD8+ T-cells that persisted long-term (〉200 days), rapidly cleared MCMV-antigen pulsed target cells, and significantly reduced MCMV replication in vivo. Since severely immunocompromised BMT recipients could be safely vaccinated with KBMA-Lm-MCMV immediately after transplant without mortality, significant levels of virus-specific CTLs could be reconstituted within 7 days of transplantation. Given the resulting high levels of durable antigen-specific lytic activity, this approach could represent a broadly applicable alternative to adoptive immunotherapy to prevent viral disease after transplantation.

Beschreibung: Background Allogeneic bone marrow transplant (BMT) is the only curative method for a number of monogenic blood disorders, including various forms of hemoglobinopathies and severe combined immunodeficiencies. Aside from the significant hurdle of finding an identical HLA-matched related donor, allogeneic BMT recipients require chronic immunosuppression to mitigate the significant risk of GVHD and are at greater risk for graft failure. Autologous gene modified HSPC potentially provide a much safer alternative to allogeneic BMT and abrogate the need for finding HLA-matched donors. Here, we report the development of a highly efficient process for generating gene modified human HSPC at clinical-scale (〉150 x 10^6 cells for 2x10^6/kg) using clinical-grade equipment. Methods Healthy donors were administered Neupogen® (10mg/kg/day) for 4-5 consecutive days and then apheresed. Enrichment of CD34+ cells was performed with the Miltenyi CliniMACS® system. Genome editing was achieved via the introduction of mRNA encoding two engineered zinc finger nucleases (ZFN) using a scalable electroporation device. Cells were harvested and cryo-preserved with a controlled-rate freezer. Cell recovery and viability, gene modification efficiency, stem cell pluripotency and engraftment potential were evaluated by in vitro assays and in a humanized NSG mouse model. Results Enrichment of CD34+ cells from mobilized leukopak products was highly efficient with the Miltenyi CliniMACS® system (median recovery = 327 million CD34+ cells per 10L mobilized leukopak). The positively selected fractions were 〉98% CD34+ by FACS analysis. Two large scale electroporation devices (BTX AgilePulse Max® and MaxCyte GT®) were evaluated. Each device is capable of electroporating up to 300 million cells. The optimal transfection conditions for both devices were first identified by using a GFP mRNA to evaluate transfection efficiency by flow cytometry, which resulted in the identification of conditions (voltage and duration) that yielded gene transfer efficiencies of 〉90%. Compatibility of this protocol with driving endogenous gene modification was evaluated using mRNA encoding ZFNs that target various endogenous gene loci. Highly efficient levels of genome editing were observed in CD34+ HSPC each transfected with a different pair of ZFNs (median = 53%, 43%, 45% and 42% modified alleles at four distinct disease-relevant loci). At the optimal mRNA dose for each ZFN pair, cell viability post electroporation was 〉80%, comparable to untransfected controls. Process suitability was evaluated by in vitro colony forming cell assay. No significant differences in colony formation were observed between gene modified and untransfected control samples. The capacity of electroporated HSPC to engraft and support multi-lineage development of human hematopoietic cells was evaluated in NSG mice, and no differences were observed between the ZFN-treated and untransfected control cells. In addition, high levels of gene modification (19-28%) were detected in bulk human cells from the blood and tissues of engrafted mice, and in various sorted cell types (bone marrow CD34 and differentiated B and T cells). Conclusion We have developed a scalable process capable of deriving 〉300 million gene-modified CD34+ HSPC. This process supports high levels of ZFN-driven genome editing, is well tolerated, and causes no discernable defect in the hematopoietic potential of these cells to develop into multiple cell lineages, with high gene editing levels maintained in the differentiated progeny of the HSPC. These results support the use of gene modified autologus HSPC for the treatment of monogenic blood disorders. Disclosures: Lee: Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Ya-Li:Sangamo BioSciences: Employment. Kim:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Wang:Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Ando:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment.