ALBERT

Description: There is a growing effort in creating chiral transport of sound waves. However, most approaches so far have been confined to the macroscopic scale. Here, we propose an approach suitable to the nanoscale that is based on pseudomagnetic fields. These pseudomagnetic fields for sound waves are the analogue of what electrons experience in strained graphene. In our proposal, they are created by simple geometrical modifications of an existing and experimentally proven phononic crystal design, the snowflake crystal. This platform is robust, scalable, and well-suited for a variety of excitation and readout mechanisms, among them optomechanical approaches.

Description: Abstract 3123 In gene therapy targeting hematopoietic cells, a quantitative assessment of the risk factors underlying insertional mutagenesis is required to assess the practical value of preventive actions. Emanating from an observation of the Copeland lab (Du et al., 2005) we developed an in vitro immortalization (IVIM) assay which determines the risk of transformation of murine bone marrow cells as a consequence of insertional upregulation of Evi1 or Prdm16. These functionally related genes encode master regulators of hematopoiesis which are involved in the pathogenesis of human leukemia and insertional transformation in human gene therapy. Using our standardized conditions, the assay can detect mutants arising with a low frequency (down to 1 in a million cells), based on their rescue and expansion upon replating. The genetic lesion associated with clonal transformation is easily identified, and we can quantify not only the incidence of mutants (number of cells required to form a mutant) but also their fitness (number of subclones obtained by replating). Using the IVIM assay, our published work has revealed the following: (1) relocating gammaretroviral enhancer-promoter sequences from the LTR to an internal position of a “self-inactivating” (SIN) vector reduces the fitness of mutants, as do mutations in transcription factor binding sites or insulators that reduce the enhancer activity; (2) cellular promoters located in SIN vectors, depending on their enhancer activity, may reduce the risk of transformation below the detection limit (〉3 logs compared to standard gammaretroviral vectors); (3) the post-transcriptional regulatory element of the woodchuck hepatitis virus does not affect insertional transformation; and (4) the lentiviral integration pattern reduces the risk of insertional transformation by a factor of ∼3 compared to gammaretroviral vectors. In the meantime, the assay has been used to assess the transforming potential of new vectors developed to treat a variety of hematopoietic disorders, most notably X-SCID, X-CGD, WAS and globinopathies. Reproducibly we found that vectors containing cellular promoters reduced the risk of insertional transformation when compared to retroviral promoters, although not all cellular promoters appeared to be free of risk. The assay has also revealed major functional differences of various insulator elements, including synthetic ones designed to block enhancer-crosstalk. Testing a battery of 8 insulators that we obtained from collaborators or designed ourselves, we found that only a subset was potent enough to significantly reduce the transforming potential of a strong retroviral enhancer-promoter. Furthermore, we assessed the transforming potential of our new alpharetroviral SIN vectors (Suerth et al., JV 2010), modified to remove a residual TATA box of the LTR. When containing a retroviral internal promoter, alpharetroviral SIN vectors were ∼9-times and 3-times, respectively, less likely than the corresponding gammaretroviral and lentiviral constructs to induce strongly replicating clones. Mutants obtained with alpharetroviral SIN vector insertions in Evi1 were not only less frequent but also had a greatly reduced fitness compared to those induced by similarly designed gammaretroviral vectors. Alpharetroviral SIN vectors containing the human elongation factor 1 alpha promoter did not immortalize cells in this assay, as previously shown for gammaretroviral SIN vectors. Finally, we performed experiments to explore the mechanistic basis of the IVIM assay. Our data suggest that its principle is the selection of mutants that resist the differentiation-inducing effect of a myeloid growth factor cocktail. Therefore, variations of the cell culture conditions have a significant impact on the sensitivity of the assay, and potentially also on the spectrum of mutants that can be isolated. The established conditions typically select for upregulation of Evi1, Prdm16, or, more rarely observed, Ras -related genes. In summary, the IVIM assay quantifies the risk of insertional mutagenesis in gene therapy, related to vector sequences and integration pattern. It is specifically useful to assess the risk of insertional upregulation of Evi1 and Prdm16 via enhancer-mediated mechanisms, in myeloid progenitor cells. It thus serves as an animal replacement assay to screen for safety-enhancing vector modifications. Disclosures: Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells.

Description: Introduction Generally, CD34+ cells are used for genetic modification in gene therapy trials. CD34+ cells consist of a heterogeneous cell population with mostly limited long-term repopulating capabilities, resulting in low long-term engraftment levels in particular in those diseases in which gene modified cells lack a proliferative advantage over non-modified cells. Therefore, modifications in gene transfer vectors and gene transfer strategies are required to improve long-term clinical benefit in gene therapy patients. One particular attractive approach to solve this problem is the improvement of HSC based gene transfer by specifically targeting cells with long-term engraftment capabilities. Material and Methods We constructed lentiviral gene transfer vectors (LV) specifically targeting CD133+ cells, a cell population with recognized long-term repopulating capabilities. Targeting is achieved by pseudotyping with engineered measles virus (MV) envelope proteins. The MV glycoprotein hemagglutinin, responsible for receptor recognition, is blinded for its native receptors and displays a single-chain antibody specific for CD133 (CD133-LV). These vectors were compared to VSV-pseudotyped lentiviral vectors in in vitro and in vivocompetitive repopulation assays using mobilized peripheral blood CD34+ cells. Results Superior transduction of isolated human hematopoietic stem cell populations (CD34+CD38- or CD34+CD133+ cells) compared to progenitor cell populations (CD34+CD38+ or CD34+CD133-) could be shown using the newly developed CD133-LV. Transduction of total CD34+ cells with CD133-LV vectors resulted in stable gene expression and gene marked cells expanded in vitro, while the number of VSV-G-LV transduced CD34+ cells declined over time. Competitive repopulation experiments in NSG mice showed a significantly improved engraftment of CD133-LV transduced HSCs. At ∼12 weeks post-transplantation gene marked hematopoiesis was dominated by the progeny of CD133-LV transduced cells in 42 out of 52 transplanted animals in the bone marrow and 39 out of 45 transplanted animals in the spleen, respectively. Consistent with this data we could show that stem cell content in the CD133-LV transduced population is about five times higher compared to the VSV-transduced population using a limiting dilution competitive repopulation assay (LDA-CRU). Experiments showing proof of principle for the application of this technology for the correction of Chronic Granulomatous Disease (XCGD) using patient derived CD34+ cells are currently ongoing. Discussion In conclusions this new strategy may be promising to achieve improved long-term engraftment in patients treated by gene therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Repression of the BCL11A protein could represent a therapeutic target for beta-hemoglobinopathies, as its knock-down has been shown to induce the expression of the fetal HBG (y-globin) gene ultimately leading to increased levels of the fetal hemoglobin tetramer (HbF, a2y2). In mice, Bcl11a is a key repressor of the murine HBG homolog Hbb-y. RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via pol III promoters has been used to modulate gene expression in a variety of mammalian cell types. However, we found a negative impact of Bcl11a knockdown on hematopoietic stem cells (HSCs), limiting the repopulation efficiency and long-term engraftment after genetic modification, which is a major impediment for its translation into human therapeutic applications. To achieve lineage-specific targeting of mRNAs in an attempt to reduce HSC toxicity, expression of shRNAs via pol II promoters is required, necessitating embedding the shRNA in mammalian microRNA (shRNAmir) sequences for expression and processing. To achieve optimal knockdown of the Bcl11a transcription factor in erythroid progenitor and precursor cells, we first compared the efficiency of mRNA modulation via pol III (U6-promoter) vs pol II (SFFV-promoter) based lentiviral vectors. We demonstrate a 100-1000 fold lower Hbb-y induction using shRNAmir vs pol III mediated shRNA vector backbones due to reduced Bcl11a knockdown efficiency. In order to understand the molecular basis for these differences, small RNA sequence analysis was performed on murine erythroleukemia cells (MEL) cells transduced by multiple shRNA–shRNAmir pairs. We show that shRNAs expressed via a U6 promoter yield guide strand sequences which differ by a 2-4 nt shift compared to pol II driven (shRNAmir) mature guide strand sequences. RNA sequencing demonstrated that the stretch of uridines making up part of the pol III termination signal is transcribed and included at the 3’ end of the shRNA. This results in the generation of mature guide strand sequences with an alternative seed sequence compared to the predicted sequence and compared to miRNA embedded shRNAs. The difference in the seed sequences between the two expression systems strongly influences the efficacy of target gene knockdown, leading to reduced knockdown in pol II based vectors. We engineered a 4bp shift into guide strands of shRNAmirs that resulted in a faithfully processed shRNA sequence (a mature guide strand sequence identical to U6-driven sh-RNAs) and improved knock-down efficiency of Bcl11a at the protein level in most cases. The improved knockdown of Bcl11a was associated with a 100-300-fold enhancement of Hbb-y induction in MEL cells. Based on these results, we propose a modified strategy for the prospective design of shRNAmirs derived from shRNA screens in pol III vector backbones to achieve lineage-specific regulation of target genes. Targeted expression of shRNAmiRs to the erythroid compartment driven by a b-globin promoter/LCR element circumvented the detrimental effect on HSC engraftment, while still mediating efficient BCL11A knockdown, leading to high y-globin induction and formation of substantial amounts of fetal hemoglobin in human CD34-derived erythroid cells in vitro. Disclosures No relevant conflicts of interest to declare.

Description: Although therapeutic genome editing of autologous hematopoietic stem cells (HSCs) in principle could cure β-hemoglobinopathies, CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in HSCs. Here we demonstrate selection-free on-target editing of the BCL11A erythroid enhancer by Cas9:sgRNA ribonucleoprotein in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. First we screened a set of 20 guide RNAs targeting the functional core of the +58 BCL11A enhancer for maximal HbF induction by RNP delivery. We used SpCas9 protein with additional NLS sequences, synthetic modified sgRNA, and optimized electroporation buffer to produce 〉95% on-target indels disrupting a critical GATA1 binding site within the +58 BCL11A enhancer in CD34+ HSPCs. Clonal analysis showed that even 1 bp indels around the cleavage site were sufficient for HbF reactivation. Specificity was evaluated by CIRCLE-seq, a method to define genome-wide target sequences susceptible to RNP cleavage in vitro. Amplicon deep sequencing of 24 possible off-target sites from edited CD34+ cells did not reveal any off-target editing with limit of detection 0.1% allele frequency. Despite transient induction of a p53 transcriptional response peaking at 4-8 hours after RNP electroporation, we found no evidence of selection for TP53 or 94 other hematologic malignancy associated mutations by targeted deep sequencing. Edited CD34+ HSPCs from healthy donors contributed to multilineage engraftment of primary and secondary immunodeficient mouse recipients at similar levels as unedited control cells. Likewise we found that edited CD34+ HSPCs from a plerixafor-mobilized SCD donor contributed to marrow engraftment and multilineage hematopoiesis in immunodeficient NBSGW mice after 16 weeks at similar levels as unedited cells, with ~95% indel frequency for engrafting healthy and SCD donor cells. Edited engrafting SCD cells were similarly competent for secondary transplantation as unedited controls. Erythroid progeny of edited engrafting sickle cell disease HSCs expressed therapeutic levels of fetal hemoglobin (HbF) and resisted sickling. Erythroid progeny of edited CD34+ HSPCs from 7 transfusion-dependent ß-thalassemia donors showed restored globin chain balance and amelioration of microcytosis and poikilocytosis. We found that compared to the bulk CD34+ HSPC pool, HSCs preferentially underwent nonhomologous as compared to microhomology mediated end-joining repair based on three assays: sorting of CD34+ CD38- CD90+ CD45RA- cells; isolation of cells in G0, G1, S, and G2/M cell cycle phases; and evaluation of long-term engrafting cells in immunodeficient mice. Together these data show that NHEJ-based BCL11A enhancer editing approaching complete allelic disruption is a practicable therapeutic strategy to produce durable HbF induction in SCD and ß-thalassemia. Disclosures Esrick: Bluebird Bio: Honoraria. Williams:Bluebird Bio: Research Funding.

Description: RHOH encodes a GTPase-deficient, hematopoietic-specific small GTPase first identified as a hypermutable gene in DLBCL (Pasqualucci et al. 2001). RhoH is critical for T cell receptor signaling and Rhoh-deficient (RhohKO) mice have T cell lymphopenia (Gu et al., 2006) and loss of function mutations of RHOH are associated with Epidermodysplasia Verruciformis (Crequer et al., 2012). However, the role of RhoH in the biology of DLBCL is still unknown and its role in B lymphoid development is incompletely studied. We investigated the role of RhoH in normal germinal center formation and in a murine model of DLBCL by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice (Cattoretti G, et al., 2005). In young RhohKOmice, deficient development of CXCR5+ follicular T helper (Tfh) cells results in defective germinal center (GC) formation and impaired immunoglobulin switching in vivo. In spite of this defect in GC formation, RhohKO; Bcl-6Tg (KOTg) mouse demonstrated accelerated lymphoma progression associated with larger spleens and significantly earlier death (Log-rank test p

Description: Inflammation shapes the immune response to foreign antigens by influencing effector immune function and is dysregulated in many forms of inflammatory bowel disease and other auto-immune conditions. It is increasingly recognized that inflammation also impacts hematopoietic stem and progenitor cell (HSPC) function. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that regulates inappropriate immune responses by suppressing inflammatory programs in mature immune cells. However, the direct role of IL-10 in modulating HSPC function and in suppressing hematopoietic cell output in the context of inflammation has not previously been demonstrated. IL10Rß-deficient (Il10rb-/-) mice have an increased inflammatory cytokine profile and develop overt colitis symptoms between 4-6 months of age. We observed an expansion in the relative frequency of immunophenotypically-defined HSPCs (LSK+: Lineage-c-Kit+Sca-1+) and frequency and absolute numbers of granulocyte-monocyte progenitors (GMPs: Lineage-c-Kit+CD34+FcgRII/III+) in pre-colitic Il10rb-/- mice relative to wildtype mice (WT) (see table). Increased GMPs correlated with increased myeloid and reduced lymphoid frequency and number in the bone marrow (BM). Consistent with the expansion in the Il10rb-/-BM HSPC compartment, transplantation of a 1:1 ratio of Il10rb-/-: WT whole BM also resulted in a 2-fold higher short-term reconstitution of Il10rb-/- CD45+ cells in peripheral blood relative to WT cells. Moreover, in vitro we observed suppressed expansion of WT but not Il10rb-/- lineage- cells after IL-10 treatment, suggesting that IL10 suppresses HSPC proliferation. To determine if IL-10 signaling affects the function of hematopoietic stem cells (HSCs), we compared an equal number of purified HSCs (LSK+CD150+CD48-) from Il10rb-/- and WT mice in repopulation assays. Il10rb-/- HSCs demonstrated a 6-fold (P