ALBERT

Description: The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.

Description: Introduction Patients aged 〉60 years (y) with acute myeloid leukemia (AML) have inferior outcomes compared to those 60y with newly diagnosed AML or high-risk MDS combining pano at doses ranging from 20 mg to 60 mg given orally on days 1, 3, 5, and 8 of induction with daunorubicin (dauno) 60 mg/m2 on days 3-5 and ara-C 100mg/m2 continuously on days 3-10 (p+7+3). Patients were allowed retreatment on p+5+2, with pano on days 1, 3, and 5 at the same doses, if the nadir BM biopsy had residual leukemia. Upon attainment of CR/CRi, patients were offered a second p+7+3 or alternative consolidation including allogeneic stem cell transplantation (alloHCT). Dose-limiting toxicity was defined based on unexpected toxicities from 7+3 induction and a standard 3+3 design was utilized for dose escalation. Peripheral blood mononuclear cells were isolated on days 1-5 and HDAC expression and histone acetylation were evaluated by western blot and quantified by densitometry. Results Overall, 22 patients were treated on the PanDA protocol. Treatment was well-tolerated in all dose-cohorts and the MTD was not reached. We treated 6 patients each at the 60 mg and 50 mg dose in the dose expansion phase due to recurrent grade 1 bradycardia. The median age was 67y-60-69y (13), 70-79y (5), and 〉80y (4). Eleven patients had de novo AML, 7 had AML with myelodysplasia related changes, 2 had 2ary AML from MPD, 1 treatment-associated myeloid neoplasm, and 1 had RAEB-2. Two patients had progressed on hypomethylating agents. Thirteen (62%) had intermediate-risk cytogenetics (cyto). Amongst 8 euploid patients, 2 had FLT3-ITDmut, 1 had FLT3-TKDmut and 2 had NPM1mut as the sole abnormality. Seven (33%) patients had complex/monosomic cyto. There were no patients with favorable cyto. There were no protocol-defined DLTs. The most common grade 2-4 TEAEs were febrile neutropenia (77%), electrolyte abnormalities (64%), diarrhea (55%), rash (55%), LFT abnormalities (32%), nausea (27%) and anorexia (27%). Of note, 36% experienced asymptomatic sinus bradycardia peaking on day 6 following pano, dauno, and 5HT3 inhibitor anti-emetics. Additionally, 9% experienced new onset grade 2 atrial fibrillation. There was no evidence of myocardial dysfunction on echocardiogram following induction. SAEs reported on study were sepsis (9%), typhlitis (5%), DIC (5%), pneumonitis (5%), and bowel perforation due to mucorales (5%). There were 2 deaths (9%) during induction, one related to typhlitis and the other to sepsis. Of 20 evaluable patients, 8 (40%) achieved CR/CRi . One patient received study treatment as consolidation, 3 received intermediate/high-dose ara-C, 1 received a hypomethylating agent, 2 underwent alloHCT and 1 had no further therapy. Median overall survival was 10 mos (ITT) and 16 mos for those who achieved a CR/CRi (p=0.005). Median relapse-free survival was 10 mos, range 3-27 mos (Figure 1). Baseline expression of HDAC class 1, 2, 3 and 6 was evaluated and was not found to be associated with response to p+7+3 arguing against a direct role in p+7+3-induced cytotoxicity. However, global histone acetylation change after pano exposure (days 1-3) was significantly associated with eventual chemo-ablation at nadir (Figure 2). Ongoing studies are evaluating markers of DNA damage response and apoptosis in patients treated with PanDA vs. concurrent 7+3 controls. Conclusions The addition of pano to 7+3 induction for older patients with AML is well-tolerated and results in clinical outcomes that are favorable in our very high-risk population. The recommended dose of pano is 50 mg daily on days 1, 3, 5, and 8 when combined with 7+3 induction in future studies. Importantly, histone acetylation change following exposure to pano but not baseline HDAC expression predicts sensitivity to combined therapy. Disclosures Wieduwilt: Sigma-Tau: Research Funding; Alexion: Honoraria. Off Label Use: Panobinostat is approved for multiple myeloma. This paper reports it's use in patients with acute myeloid leukemia.. Olin:Daiichi-Sankyo: Research Funding. Logan:Pharmacyclics: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy. Damon:Atara: Consultancy; Sunesis: Research Funding; McGraw-Hill: Patents & Royalties: Book Chapter; Sigma-Tau: Research Funding. Boyer:Onyx: Speakers Bureau. Andreadis:genentech: Employment; novartis oncology: Research Funding; cellerant: Research Funding; karyopharm: Research Funding; McGraw-Hill: Patents & Royalties: Book Chapter; Pfizer: Honoraria; Pharmacyclics: Honoraria.

Description: Introduction: Allogeneic hematopoietic cell transplantation (alloHCT) has historically been reserved for younger, fit patients with hematologic malignancies. With the introduction of non-myeloablative conditioning regimens and improved supportive care, alloHCT has been increasingly offered to older adults. Objectives: To determine the association between functional status as measured by a cancer-specific comprehensive geriatric assessment (CGA) and post-transplant outcomes in an older alloHCT patient population. Methods: We conducted a prospective cohort study of patients aged 50 or older who underwent alloHCT at the University of California San Francisco between October 2011 and September 2017. A cancer-specific CGA (1) was administered prior to alloHCT, which included measures of functional status such as Lawton Instrumental Activities of Daily Living (IADL), Medical Outcomes Study (MOS) Physical Health scale, and patient-reported Karnofsky Performance Status (KPS). Post-transplant outcomes included length of hospital stay (LOS), non-relapse mortality (NRM), progression-free survival (PFS), and overall survival (OS). Results: A total of 148 patients were included in the analysis. The median age at transplant was 62 (range 50-76). Disease types included acute myeloid leukemia (43%), myelodysplastic syndrome (26%), myeloproliferative neoplasm (12%), acute lymphoblastic leukemia (10%), non-Hodgkin lymphoma (5%), multiple myeloma (1%), and other (3%); 68% received non-myeloablative conditioning. Median follow-up was 16.3 months (range 0.9-72.7 months). Median PFS and OS were 22.9 months and 47.6 months, respectively. At baseline, 39% had at least one IADL deficit, and 88% had at least one MOS Physical Health scale deficit. The mean patient-KPS was 82.4 and mean provider-KPS was 91.6; these were weakly correlated (Spearman's r=0.39, p

Description: Background Although multiple studies have shown superiority of allogeneic hematopoietic cell transplantation (alloHCT) over autologous hematopoietic cell transplantation (autoHCT) for patients with "high-risk" acute lymphoblastic leukemia (ALL), these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT. Methods We retrospectively evaluated minimal residual disease (MRD) via next-generation sequencing (NGS) (Adaptive Biotechnologies, S. San Francisco, CA) in the PBPC leukapheresis products from 32 ALL patients who underwent autoHCT. All patients had "high-risk" ALL, as defined by B-lineage disease with WBC at diagnosis 〉30,000/uL; high-risk cytogenetics including t(9;22), t(4;11), other 11q23 abnormalities, or monosomy 7; or primary refractory disease. Peripheral hematopoietic cell mobilization consisted of cytarabine 2000mg/m2 IV every 12 hours (16,000mg/m2 cumulative) concurrent with etoposide 40mg/kg cumulative by continuous IV infusion over 4 days. The autoHCT conditioning consisted of 1320cGy total body irradiation (TBI) given in 11 fractions from day -8 to -5, etoposide 60mg/kg IV on day -4, and cyclophosphamide 100mg/kg IV on day -2. Tyrosine kinase inhibitors (TKI) were allowed for patients with Philadelphia chromosome-positive (Ph+) B-ALL. Seven patients (22%) participated in a multi-institutional trial in which the PBPC graft underwent ex vivo complement-mediatedpurging using monoclonal antibodies against CD9/CD10/CD19/CD20 for patients with B-lineage disease, or against CD2/CD3/CD4/CD5/CD8 for patients with T-lineage disease. Kaplan-Meier curves were generated using GraphPad Prism (GraphPad Software, La Jolla, CA) and statistical differences assessed via Log-Rank and Wilcoxon analyses, with a p-value of

Description: The human β globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult β globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the β globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from γ to β globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human β globin mRNA levels between lines. Human β globin mRNA levels were twofold to fourfold lower than that of mouse βmaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the β globin locus.

Description: Advances in the design and efficiency of gene delivery vectors have enabled the initiation of clinical trials in gene therapy for genetic and other disorders. However, the development of inhibitory immune responses to vector antigens and to therapeutic proteins remains an obstacle. Efforts to limit these immune responses by immunosuppressive and immuno-modulatory approaches have met with limited success. Our approach is to deliver and express viral vectors early in immune ontogeny and thereby induce immune tolerance to both vectors and therapeutic proteins. We have previously shown that in utero delivery of AAV-2 vectors produces lifelong gene expression without immune responses, and that augmented levels of gene expression are achieved with re-administration of AAV vectors. Because fetal injections are limited by technical issues, our current focus is to use a neonatal model for defining the critical period when tolerance to vector and transgene may be achieved by primary injection. We are also exploring mechanisms of tolerance induction to neo-antigens. We have delivered AAV serotypes 1 and 8 with higher transduction efficiencies than AAV-2, to assess the expression levels, duration, and tissue distribution of luciferase by semi-quantitative longitudinal in vivo bioluminescence assays. In both C57BL/6 and BALB/c strains, neonatal injection of AAV1-Luc or AAV8-Luc by either IP, IV or IT routes produces lifelong gene expression. After IP injection at day 1–2 of life, gene expression increases 10–20 fold over the next several months. Highest levels of expression were achieved by IP injection, with lowest levels observed after IV injection. Injection of AAV1-Luc achieved higher levels of luciferase expression than did injection of AAV8- Luc. In contrast to the localized distribution of AAV1 mediated luciferase expression in the injected area, widespread, systemic expression of luciferase mediated by AAV8 after neonatal delivery is observed, regardless of the route of delivery. The effect of this altered tropism on gene expression levels and tolerance induction is being examined. In both C57BL/6 and BALB/c mice, IP injection of AAV1-Luc or AAV8-Luc at 1–2 days, 1 week, 2 weeks, or 3 weeks of age produced lifelong expression of luciferase and resulted in increasing levels of antibody responses against AAV1 or AAV8 with increasing age at primary injection. Antibody titers to AAV1 or AAV8 in animals injected at day 1–2 of life were comparable to background levels in uninjected animals. In C57BL/6 mice receiving a primary injection of AAV8-Luc, secondary injection of AAV8-Luc boosted the antibody response to AAV8 in the animals first injected at 1 week, 2 weeks or 3 weeks, but not in the animals injected at 1–2 day of life. We are currently exploring whether augmented expression with re-administration of AAV vectors in adult animals is due to an active process such as tolerance, partial tolerance or anergy. Developing strategies for the induction of tolerance to gene delivery vectors and therapeutic gene products will be an important advance for gene therapy for genetic and other disorders.

Description: The human β globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult β globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the β globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from γ to β globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human β globin mRNA levels between lines. Human β globin mRNA levels were twofold to fourfold lower than that of mouse βmaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the β globin locus.

Description: Transplantation of genetically engineered hematopoietic stem cells (HSC) holds promise for treatment of genetic and other disorders; however, several obstacles restrict the clinical application of this approach. These include leukemogenesis resulting from insertional mutagenesis, the lack of a competitive advantage for transplanted genetically engineered autologous HSCs, and the requirement for the construction and clinical approval of a gene transfer vehicle specific to each disorder. Allogenic transplantation approaches are further limited by a shortage of compatible donors, the toxicity of preparative regimens, immunosuppression, and graft versus host disease (GVHD). We have established a neonatal transplantation model to develop strategies to overcome these hurdles to HSC transplantation and gene therapy. We have uniquely combined allogenic transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of tolerance induction mechanisms to neo-antigens, and allogenic stem cell engraftment during immune ontogeny. In this model, bone marrow-derived HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K) and GFP (MAG vector). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. This enables enrichment of donor cells at the stem cell level by in vivo chemoselection. Furthermore, in vivo administration of BG/BCNU may potentially deplete allo-reactive cells of donor and host origin, reducing the need for toxic ablative or immunosuppressive treatment. The in vivo selection strategy was first tested in neonates in the absence of MHC barriers. Syngenic whole bone marrow (WBM) was transduced with MAG and 5 × 105 cells were transplanted. Five weeks after transplantation and prior to chemoselection, 0.5% of cells expressed GFP in peripheral blood. Following two cycles of in vivo selection we observed 39.5% expression of GFP in peripheral blood. To assess the engraftment of fully MHC-mismatched HSC in neonates without in vivo selection, 107 B10.Br (H-2k) WBM was transplanted into C57BL/6 (H-2b) neonates using a non-myleoablative regimen. This resulted in 98% engraftment in adults without development of GVHD. To evaluate in vivo selection in an allogenic setting, 2 × 106 MAG transduced WBM from BALB/c (H-2d) adult mice were transplanted into C57BL/6 × BALB/c F1 (H-2b/d) neonatal mice. Despite having a graft versus host MHC disparity, we observed an increase from 4.7% (5 weeks after transplant) to 37.4% GFP expression following two cycles of in vivo chemoselection without evidence of GVHD. We have demonstrated successful engraftment and enrichment of both syngenic and allogenic HSC expressing MGMTP140K following neonatal transplantation and in vivo selection. Stable engraftment was achieved without myeloablation or post-transplant immunosuppression. No evidence of GVHD was observed after full MHC-mismatched transplantation. These results hold promise for the development of non-myeloablative allogenic transplant approaches using this in vivo selection strategy.