Byoung Y. Ryu

Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

229. PGE2 Increases Lentiviral Vector Transduction Efficiency of Human HSC

Gene therapy for congenital hematopoietic disorders frequently relies on ex vivo lentiviral transduction of isolated CD34+ hematopoietic progenitor cells. Through a high-throughput small molecule screen, we identified PGE2 as a positive mediator of lentiviral transduction of hematopoietic stem and progenitor cells enriched from mobilized peripheral blood (PB CD34+ cells). CD34+ cells transduced with a VSVG-pseudotyped lentiviral vector in the presence of cytokines and 10 uM PGE2 yielded a vector copy number per cell (VCN) approximately 2-fold higher than CD34+ cells transduced in the absence of PGE2. This effect was seen consistently in 16 of 16 tested normal human donors in vitro, as well as primary CD34+ cells from both thalassemia and sickle cell disease patients. Importantly, PGE2 was observed to improve transduction of prospectively-isolated CD34+CD38- hematopoietic stem cells - a sub-population thought to be enriched for the long term repopulating stem cell. Transduction improvements were not associated with increased viral entry, but were associated with elevated expression of cAMP genes, supporting a post-entry mechanism of action that involves cAMP signaling downstream of prostaglandin receptors. Lastly, in a mouse xenotransplantation model of hematopoietic stem cell transplant, transduction of PB CD34+ cells in the presence of PGE2 improved VCN levels in engrafted human CD45+ cells 4-5 months post-transplant by ~2-fold without adversely affecting overall human cell engraftment. These data suggest that PGE2-mediated improvements in lentiviral transduction of human CD34+ cells could result in higher transduction efficiency and provide potential benefit in clinical gene therapy applications.

Robust erythroid differentiation system for rhesus hematopoietic progenitor cells allowing preclinical screening of genetic treatment strategies for the hemoglobinopathies

BACKGROUND AIMS γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. METHODS We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. RESULTS Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). DISCUSSION In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.

458. Development of a Stable Producer Cell Line for Scalable Lentiviral Vector Production for Gene Therapy of Hemoglobinopathie

Current manufacturing of clinical grade lentiviral vectors (LVVs) for gene therapy applications commonly relies on transient transfection of adherent 293T cells. Improvements in production efficiency and scalability would provide value in meeting the needs for increased amounts of vector required for clinical development. An inducible producer cell line grown in suspension culture represents a potentially more scalable manufacturing process for LVV production which eliminates the need for costly plasmid and transfection reagents. We have engineered a packaging cell line by introducing doxycycline-inducible Gag-Pol, Rev, and VSVG envelope genes into a suspension cell line. Packaging cell clones were isolated by single cell sorting and screened by qPCR for the presence of the delivered genes. Virus production was assessed by transient transfection of a lentiviral vector and doxycycline treatment. A titer of up to 5.0E6 transduction units per milliliter (TU/mL) was observed with packaging cell line stability demonstrated after greater than four months of continuous passage. Next, a self-inactivating lentiviral vector was excised from its plasmid backbone and ligated in vitro to a linear neomycin resistance cassette and used to transfect the lentiviral packaging cell line to generate a panel of producers. Following two weeks of G418 drug selection, adherent colonies were plucked and screened for viral production followed by single cell sorting to isolate individual producer clones. From five different plucked colonies with titers greater than 3.0E6 TU/mL, more than 100 clones were screened and 7 were identified that produced a harvest titer greater than 1.0E7 TU/mL. Four of these clones were successfully re-adapted to suspension and scaled up to 3L culture. LVV generated from individual producer clones was compared for the ability to transduce adult mobilized CD34+ hematopoietic stem cells (HSCs). Interestingly, the vector copy number (VCN) in CD34+ HSCs for the LVV derived from the producer cells varied between clones. Clones that produced the highest VCN have been chosen for further characterization in order to better understand the differences in HSC transduction. These lentiviral producer cell lines represent an important first step toward the creation of a manufacturing process that can better support clinical and commercial development of HSC lentiviral gene therapy.