Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product.

INTRODUCTION: In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαß/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαß/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. METHODS: We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαß+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. RESULTS: Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαß+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation. CONCLUSION: The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαß/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

Next generation FIX muteins with FVIII-independent activity for alternative treatment of hemophilia A.

BACKGROUND: FVIII neutralizing antibodies are the main complication of substitution therapy in hemophilia A (HA); auto-antibodies against FVIII causing acquired HA can also occur. Treatment of inhibitor patients remains challenging because prophylactic treatment with existing FVIII bypassing agents, all based on constitutively active coagulation factors, is difficult due to their short half-life. OBJECTIVES: To generate zymogenic FIX variants with FVIII-independent activity for gene- and protein-based therapy for HA. METHODS: Modifications were introduced into FIX based on current knowledge of FIX structure and FVIII-independent function followed by random screening. Activity, thrombin generation and FX activation by FIX mutants were characterized in the presence and absence of FVIII. Phenotype correction of promising candidates was assessed by the tail-clip assay in FVIII-knockout mice. RESULTS: About 1600 clones were screened and three mutations (L6F, S102N and E185D) identified, which improved FVIII-independent activity in combination with our previously described variant FIX-ITV. By systematic combination of all mutations, six FIX mutants with the desired bypassing activity were designed. Candidate mutants FIX-IDAV and FIX-FIAV demonstrated the most efficient thrombin generation in FVIII-deficient plasma and had considerably increased activities towards FX in the absence of FVIII, in that they showed an up to 5-fold increase in catalytic efficiency. Expression of FIX-IDAV in FVIII knockout mice reduced blood loss after the tail-clip assay, even in the presence of neutralizing FVIII antibodies. CONCLUSION: Activatable bioengineered FIX molecules (as opposed to pre-activated coagulation factors) with FVIII-independent activity might be a promising tool for improving HA treatment, especially for patients with inhibitors.

Oral gene therapy for hemophilia B using chitosan-formulated FIX mutants.

BACKGROUND: Oral gene delivery of non-viral vectors is an attractive strategy to achieve transgene expression. Although expected efficacy from non-viral delivery systems is relatively low, repeated vector administration is possible and may help to obtain durable transgene expression in a therapeutic range. OBJECTIVES: To test the principle feasibility of using factor (F) IX variants with improved function combined with an optimized oral delivery system in hemophilia B (HB) mice. METHODS: FIX modifications were introduced by site-directed mutagenesis into plasmid- or minicircle-based expression cassettes. Vectors were formulated as chitosan nanoparticles for oral delivery to HB mice. Protection of vector DNA in nanoparticle constructs and transfection efficiency were characterized. HB mice received eGFP-formulated chitosan nanoparticles to confirm gene transfer in vivo. FIX expression, phenotype correction and the potential of nanoparticles to induce immunotolerance (ITI) against exogenous FIX were evaluated after repeated oral administration. RESULTS: Transfection of HEK 293T cells or livers of FIX-knockout mice with nanoparticles resulted in GFP or functional FIX expression. Oral administration of FIX mutants resulted in exclusive FIX expression in the small intestine, as confirmed by RT-PCR and fluorescence staining. HB mice demonstrated transient FIX expression reaching > 14% of normal activity and partial phenotype correction after oral delivery of FIX mutants with high specific activity and improved tissue release. CONCLUSION: The feasibility of oral, non-viral delivery of FIX was established and improved by bioengineered FIX proteins and optimized vectors. Thus, these data might point the way for development of a clinically applicable oral gene transfer strategy for hemophilia B.