ALBERT

Description: Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Description: Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Description: Abstract 1589 Human induced pluripotent stem cells (iPSCs) that are functionally similar to embryonic stem cells (ESCs) hold great potential for cell and gene therapies, disease modeling and drug development. The earliest success was achieved by using adherent fibroblastic cells and retroviral vectors that transduce fibroblasts very efficiently. It is also highly desirable to reprogram postnatal blood cells, including those from cord blood (CB) and adult peripheral blood (PB), which are easily accessible and less exposed to environmental mutagens. In 2009, we and others have achieved the reprogramming of human postnatal blood cells using the 4 Yamanaka factors delivered by retroviral vectors. We also found that reprogramming efficiencies of CB and PB CD34+ cells are higher than age-matched fibroblasts or MSCs. This may result from an epigenetic profile of hematopoietic CD34+ cells that appears closer to iPSCs/ESCs than that of fibroblasts/MSCs to iPSCs/ESCs. To generate integration-free iPSCs that produce hematopoietic progeny efficiently, we attempted to reprogram adult PB as well as CB cells by OriP/EBNA1 episomal vectors, which were used previously to reprogram foreskin fibroblasts albeit at a low efficiency (Yu/Thomson, 2009). When one of the best combinations (#6, 3 plasmids) was used, 1–3 candidate iPSC clones per 1 million cells were obtained as reported (Yu/Thomson, 2009). We and others found that the efficiency of generating iPS clones was even lower with human adult somatic cells by the 3 vectors. To improve, we constructed a new episomal reprogramming vector system using 1–2 OriP/EBNA1 plasmids. One (pEB-C5) expresses 5 factors (OCT4/SOX2/KLF4/Myc/LIN28), and the second expresses SV40 T antigen (Tg). CB and adult PB CD34+ cells were first cultured for 4 days and expanded ≥4-folds. The expanded cells (1 million) were then transfected once by the 1–2 new OriP/EBNA1 plasmids we constructed. Fourteen days later, we obtained on average 250 and 9 TRA-1-60+, iPSC-like colonies from CB and adult PB cells, respectively, when both pCB-C5 and pEB-Tg were used. A single plasmid (pEB-C5) can also generate iPSCs although the efficiency is ∼4-folds lower. Five characterized iPSC lines derived from CB and adult PB CD34+ cells (with or without Tg) are karyotypically normal and pluripotent. After successful reprogramming and expansion, episomal DNA is gradually lost in proliferating iPSCs. After serial expansions for 11–12 passages, vector DNA was undetectable either as episomes or in the genome of the 5 iPSC lines. We next extended this approach to reprogram un-fractionated adult PB mononuclear cells (PBMCs) including those from a sickle cell patient (SCDB003). To achieve better cell proliferation that is critical to iPSC production, we used a culture condition that favors the formation and proliferation of erythroblasts from PBMCs. PBMCs purified by standard Ficoll gradient were cultured in a serum-free condition with cytokines SCF, EPO and IL-3. Although cell death was observed and cell number decreased significantly in the first 4 days, equal or more cells than input were obtained by day 8. The expanded cells morphologically resemble pro-erythroblast cells, and express high-level CD71. Less than 1.5% of them express markers of T cells (CD3, CD2, CD4 and CD8) and B cells (CD19 and CD20). When 2×106 expanded SCDB003 cells (achievable from PBMCs in 1 ml or less PB) were transfected by the 2 OriP/EBNA1 plasmids and reprogrammed in the presence of butyrate, we observed 8 colonies at day 14 that are TRA-1-60+ and iPSC-like. The second plasmid (pEB-Tg) was not essential although it enhanced the efficiency by ∼4 folds. We picked and characterized 3 iPSC-like colonies derived from PBMCs with or without Tg. All of them express pluripotency markers and behave as typical iPSCs. So far we do not have evidence if they are derived from committed T or B cells that somatic mutations altered and rearranged their genomes. We are currently examining karyotypes, in vivo pluripotency, and status of episomal vectors in 3 PBMC-derived iPSCs. As compared to recent studies using viruses that preferentially reprogram human T cells with a rearranged genome, our method of using 1–2 plasmids is virus-free and genomic alteration-free. The ability to obtain integration-free human iPSCs from a few ml PB by 1–2 plasmids will greatly accelerate uses of iPSCs in both research and future clinical applications, epically for blood disease modeling and treatment. Disclosures: No relevant conflicts of interest to declare.

Description: RNA-guided Cas9 nucleases based on the prokaryotic CRISPR-Cas systems provide an unprecedented ease in being able to edit the genomes of diverse organisms. As a single unifying factor capable of co-localizing RNA, DNA, and protein, I believe tools and techniques based on Cas9 will grant exquisite control over cellular organization, regulation, and behavior. Here I will describe work on development of the CRISPR-Cas9 targeting methodology, and detail current and prospective genome-engineering methodologies. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 812 Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells such as postnatal fibroblasts to iPS cells that resemble adherent human embryonic stem (hES) cells. It is also highly desirable to reprogram blood cells that are easily accessible and less exposed to environmental mutagens. The large numbers of umbilical cord blood (CB) cells that are collected and stored in multiple cell banks are examples that could be used as a source of either autologous or allogeneic but histo-compatible iPS cell lines. As in vitro expansion of hematopoietic stem/progenitor cells from CB and adult sources remains a challenge, unlimited expansion of derived iPS cells in combination with further optimized hematopoietic differentiation methods should provide a vital alternative to amplify histo-compatible blood stem cells for blood/BM transplantation purposes. More critically, the ability to reprogram blood cells is essential if one wishes to generate iPS cells containing somatic mutations that are restricted to the blood cells and found in acquired hematological disorders, such as myeloproliferative disorders (MPDs), in order to investigate their pathogenesis. The BCR/ABL-negative MPDs, which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are a heterogeneous group of diseases characterized by increased proliferation of erythroid, megakaryocytic and myeloid lineages alone or in combination. The acquired common somatic mutation JAK2-V617F is present in 〉95% of PV, and ∼50% of ET and PMF patients. The discovery of this mutation in 2005 has significantly improved our understanding of MPD mechanisms and has intensified the search for drug(s) that may effectively inhibit this aberrant kinase activation. However, new disease models are needed to answer questions it raised such as how gene dosages of JAK2-V617F and other pre-disposing mutations affect the MPD pathogenesis. Here we report the derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for hematopoietic differentiation and disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood, adult peripheral blood and marrow CD34+ cells of healthy donors. The hematopoietic differentiation potential of these human iPS cells was examined by an improved method of EB formation and differentiation under a feeder- and serum-free condition. After two weeks of treatment, the cells were harvested and analyzed by both hematopoietic colony-forming assays and FACS for the presence of hematopoietic markers. Both myeloid and erythroid colonies were detected as we have observed using hES cells. By FACS analysis, CD45+ (27%-64%) and CD43+ (36%-60%) hematopoietic cells co-expressing undetectable to intermediate levels of CD34 marker were also observed. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of two patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype and pluripotency. To determine if these MPD iPS cell lines could be used as a model to study abnormal human hematopoiesis, we used the same serum-free differentiation protocol to direct them into hematopoietic lineages. Similar to the increased erythropoiesis of hematopoietic progenitor (CD34+) cells isolated from PV patients, including one subject whose blood-derived iPS cells were used in this study, re-differentiated hematopoietic progenitor (CD34+CD45+) cells from the PV-iPS cells showed enhanced erythropoiesis as compared to those from the iPS cells derived from normal CD34+ cells. They also showed a gene expression pattern similar to the primary CD34+ cells from the PV patient. These iPS cells thus provide a renewable cell source and a prospective model for investigating MPD pathogenesis. In combination with the gene targeting technology we recently described, human iPS cell lines derived from patients and subsequent hematopoietic differentiation technologies provide a novel model for investigations of various blood diseases with either acquired or inherited mutations. Disclosures: No relevant conflicts of interest to declare.

Description: The aberrant and constitutive activation of the HOXA cluster genes and the their-co-factor MEIS1 (HOX/MEIS) is a recurrent feature in several types of myeloid and lymphoid leukemias. Aberrant HOX/MEIS expression has been shown to drive limitless leukemia stem cell self-renewal and is therefore an attractive target for therapy in acute myeloid leukemia (AML). However, since HOX/MEIS genes encode DNA-binding transcription factors, small molecules targeting these proteins directly are lacking. Furthermore, targeting the HOX/MEIS network is complicated by the fact that these genes are coordinately regulated and have redundant functions in sustaining leukemic self-renewal. One way of therapeutically targeting aberrant HOX/MEIS transcription is the identification and pharmacologic inhibition of upstream chromatin regulators that coordinately modulate their expression. In order to identify such chromatin regulators, we made use of an endogenous GFP reporter knocked-in to the MEIS1 locus in the high HOX/MEIS-expressing U937 human AML cell line. Using this system, we first performed a high-throughput flow-cytometry-based small-molecule inhibitor screen with a library of 261 compounds targeting epigenetic regulators. In our screen, the most potent hits that reproducibly showed 〉50% MEIS1-GFP inhibition were small molecules that targeted DOT1L, the histone methyltransferase. DOT1L inhibitors have already been well-characterized as HOX/MEIS regulators and most epigenetic regulators are not targeted by existing compound libraries. Therefore, we decided to use a genetic screening approach to more extensively interrogate the landscape of epigenetic regulators of HOX/MEIS expression in AML. For this, we designed a custom computational pipeline and built a CRISPR library of 10,000 sgRNAs targeting functionally conserved protein domains of all catalogued chromatin modulatory proteins (〉 600 proteins - 5 sgRNAs per conserved domain). This list of epigenetic regulators included histone modifying enzymes, chromatin readers, nucleosome remodelers, adaptor proteins and proteins involved in DNA and RNA modifications, as well as other transcriptional regulators. Using this comprehensive, domain-focused CRISPR library, we conducted a phenotypic enrichment screen. Specifically, we used flow cytometry to purify the top 20% GFP-MEIS1 (high) and bottom 20% GFP-MEIS1 (low) expressing cells and identified sgRNAs that were enriched particularly in the GFP-MEIS1 -low vs -high fraction using next generation sequencing. Given the extent and complexity of the CRISPR library, our approach uncovered members of six distinct chromatin modifying complexes as MEIS1 regulators (MAGeCKFlute pipeline, 2 SD 〉 mean) and we could validate 〉 10 of these hits as bonafide regulators of MEIS1 as well as HOXA genes. We also demonstrated their essentiality for the proliferation of HOX-driven AML cells using arrayed sgRNA competition assays. These validated hits included several known as well as novel chromatin readers and writers amenable to small-molecule targeting. We focused our attention on the KAT7/JADE3 complex and the casein kinase 2 (CK2) family that we validated as potent and selective regulators of HOX/MEIS expression in AML cells. Our studies demonstrated that genetic depletion of components of the KAT7 complex or of the CK2 family could reverse HOX/MEIS activation in human AML cells, leading to a progressive loss of proliferative potential. Importantly, the use of the clinical-grade CK2 inhibitor CX4945 (Silmitasertib) caused a concentration-dependent down-regulation of HOX/MEIS expression in models of HOX-driven AML, leading to significant anti-leukemia effects. Our study provides a framework for the multiplexed identification of actionable dependencies targeting therapeutically recalcitrant oncogenic networks in cancer. Specifically for AML, since Silmitasertib is in Phase 2 trials for treatment of other cancers, our studies may solve the long-standing problem of targeting leukemia stem cells in AML potentially overcoming therapy refractoriness in this devastating disease. Disclosures No relevant conflicts of interest to declare.