Hereditary Persistence Of Fetal Hemoglobin Mutation Research Articles

In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.

Individuals with β-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in > 60% -113 A>G conversion, with 30% γ-globin of β-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from β-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.

In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice

AbstractBase editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over β-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the –113A>G HPFH mutation. In a β-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human β-globin. The average –113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage–negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of β-hemoglobinopathies.

Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T>C, -175 T>C and -113 A>G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were <2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T>C), 60±10% (-175 T>C), and 42±7% (-113 A>G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T>C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P>0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T>C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

In Vivo HSC Gene Therapy with Base Editors Allows for Efficient Reactivation of Fetal Globin in Beta-Yac Mice

Base editors are capable of installing precise nucleotide mutations at targeted genomic loci and present the advantage of avoiding double-stranded DNA breaks. Here, we aimed to target critical motifs regulating gamma-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully rescued a panel of cytidine and adenine base editors (CBE and ABE) targeting the BCL11A enhancer or recreating naturally occurring Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the HBG1/2 promoter. In HUDEP-2 cells, all five tested vectors efficiently installed target base conversion and led to gamma-globin reactivation. We observed significant gamma-globin protein production (~23% over β-globin) by using an ABE vector HDAd-ABE-sgHBG#2 specific to the -113A to G HPFH mutation in HBG1/2 promoter. This vector was therefore chosen for downstream in vivo hematopoietic progenitor/stem cell (HSPC) transduction studies in mice that carry 248kb of the human β-globin locus (β-YAC mice) and thus accurately reflect globin switching. An EF1a-mgmtP140K expression cassette flanked by frt and transposon sites was included in the vector for allowing in vivo selection of transduced cells. After in vivo HSPC transduction with HDAd-ABE-HBG#2 + HDAd-SB and low doses of chemoselection, an average of over 40% HbF-positive cells in peripheral red blood cells was measured. This corresponded to ~21% gamma-globin production over human β-globin. The -113 A to G conversion in total bone marrow cells was on average 20%. Compared to untransduced mice, no alterations in hematological parameters, erythropoiesis and bone marrow cellular composition were observed after treatment, demonstrating a good safety profile of our approach. No detectable editing was found at top-scored potential off-target genomic sites. Bone marrow lineage-negative cells, isolated from primary mice at week 16 after transduction, were capable of reconstituting secondary transplanted mice with stable HbF expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic 4.9kb deletion and no detectable toxicity in human CD34+ stem cells. Our observations demonstrate that base editors delivered by HDAd5/35++ vectors represent a promising strategy for precise in vivo genome engineering for the treatment of hemoglobinopathies. Disclosures Lieber: Ensoma, Inc: Consultancy, Research Funding.

EDIT-301: An Experimental Autologous Cell Therapy Comprising Cas12a-RNP Modified mPB-CD34+ Cells for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. Fetal hemoglobin (HbF) is a major modifier of SCD severity. Studies showed that individuals with compound heterozygosity for sickle hemoglobin (HbS) and hereditary persistence of fetal hemoglobin (HPFH) that expressed approximately 30% HbF did not show features of SCD. Therefore, we are developing EDIT-301, an experimental autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a RNP (ribonucleoprotein) to promote HbF expression to treat SCD. Several HPFH mutations have been reported at the HBG locus. In particular, disruption of the distal CCAAT-box region of the HBG1/2 promoters was associated with elevated levels of HbF, suggesting that this region is a relevant genome editing target for the treatment of SCD. Genotype to phenotype analysis at the distal CCAAT-box region of the HBG1/2 promoters identified the mutations leading to elevated HbF expression. Indels disrupting more than 3 nucleotides were generally associated with elevated HbF expression while smaller indels had lower to no impact. We evaluated editing at this site using several RNP configurations, based on either SpCas9 or Cas12a (also known as Cpf1). Cas12a RNP resulted in larger deletions and a higher frequency of productive indels than SpCas9 RNP. Furthermore, productive indels generated with SpCas9 RNP relied predominantly on microhomology mediated end joining (MMEJ) mechanism. As the MMEJ repair mechanism is not frequently used by hematopoietic stem cells (HSC), productive editing may be low in vivo. In contrast, Cas12a RNP generated more productive indels at the HBG1/2 promoters target site irrespective of the DNA repair mechanism. We therefore postulated that editing at the distal CCAAT-box region with Cas12a would better support long-term persistence of productive indels and robust HbF expression. Consistent with this hypothesis, 80-90% editing was observed after electroporation of mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from healthy donors with a Cas12a RNP targeting the HBG1/2 promoters, resulting in approximately 40% of HbF expression in their erythroid progeny. Infusion of the modified CD34+ cells into NBSGW mice resulted in long-term multi-lineage reconstitution at 16 weeks post-infusion, with no reduction in editing levels compared to those at the time of infusion. A diverse on-target editing profile was observed in all animals, indicative of a polyclonal engraftment. Erythroid cells purified from the bone marrow of animals that received Cas12a-RNP treated cells demonstrated robust HbF expression averaging 40-50% compared to ~5% observed in the vehicle-treated control group. Off-target activity of the Cas12a RNP was also evaluated. A set of candidate off-target sites was first determined using orthogonal methods, including: in-silico prediction, Digenome-Seq and GUIDE-Seq. Each candidate site was then analyzed for editing by targeted PCR-NGS in electroporated CD34+ cells. No off-target editing was verified, demonstrating the specificity of this Cas12a RNP. Taken together, we identified a specific Cas12a RNP that efficiently edited the distal CCAAT-box region at the HBG1/2 promoters in CD34+ cells. These edited CD34+ cells led to long-term polyclonal multilineage engraftment and therapeutically meaningful levels of 40-50% HbF in vivo. Based on these results, IND-enabling activities have been initiated for EDIT-301: an experimental autologous cell therapy comprising Cas12a-RNP modified mPB-CD34+ cells for the potential treatment of SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Sousa:Editas Medicine Inc.: Employment, Research Funding. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Scott:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Capehart:Editas Medicine Inc.: Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Fernandez:Editas Medicine Inc.: Employment, Equity Ownership. Myer:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Chang:Editas Medicine Inc.: Employment, Equity Ownership.

Combining HPFH Mutations in Human Adult HSCs to Enhance Reactivation of Fetal Hemoglobin

Despite the significant advances in developing novel therapies for hemoglobinopathies, developing a universal and robust therapeutic approach, able to achieve an event-free result in all β0/β0 thalassemic and sickle cell patients is a work in progress. It is known that increase of Fetal hemoglobin (HbF) can lead to an ameliorated clinical picture up to transfusion independency. Inactivation via genome editing of γ-globin suppressors or introduction of Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the promoter of hemoglobin gamma (HBG) have been shown to significantly increase the endogenous HbF expression. Here we studied the effect in HbF reactivation by targeting the binding site of the two main HbF silencers, BCL11a and LRF and how it compares to the well-established editing of the BCL11a erythroid enhancer. Furthermore, in order to achieve higher levels of fetal hemoglobin, we assayed the effect of all combinations of these cis and trans acting mutations. We first observed that all single knock outs had similar effects in HbF reactivation. Interestingly, by targeting both sites in the gamma globin promoter we observed a decrease in HbF induction compared to each single edit, suggesting a possible binding of a gamma globin activator in the in-between region. However, editing of the BCL11a erythroid enhancer and either of the two silencer binding sites yielded maximum levels of fetal hemoglobin, with pancellular HbF expression, both in vitro and in vivo following xeno-transplantation in NSGW mice. Collectively, our data suggest that the combination of two fetal globin reactivation mutations, one cis and one trans, have the potential to significantly increase HbF both totally and on per cell basis. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

FUNCTIONALLY DISTINCT UTILIZATION OF CIS-REGULATORY ELEMENTS IN HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

Hereditary persistence of fetal hemoglobin (HPFH) mutations are rare, naturally-occurring variations which reduce the severity of beta-hemoglobinopathies by increasing postnatal expression of the gamma-globin genes (HBG1/2). Genome editing to inhibit erythroid transcriptional silencers or alter cis-elements in the HBG1/2 promoters recapitulates HPFH and should be tractable clinically. To define mechanisms involved with reactivation of fetal hemoglobin (HbF), we performed Cas9-mediated homology-directed repair to introduce HPFH mutations at the -115 distal CCAAT box cluster of the HBG1/2 promoters in an immortalized human erythroid cell line. HPFH mutations at positions -117, -114, -113, -110, and a 13-nucleotide (nt) deletion significantly elevated HbF levels compared to controls. Previous data suggest HPFH mutations either block repressor binding or create new binding sites for activators, such as GATA1. Using Cleavage Under Targets and Release Using Nuclease (CUTR and 2) an intact proximal CCAAT box is required for HBG1/2 de-repression by HPFH mutations in the distal CCAAT box. Elucidating the HBG1/2 cis-elements that control the perinatal gamma-to-beta globin switch should facilitate the development of optimal precision medicine-based treatments for beta-hemoglobinopathies.

A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site

Abstractβ-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations in the adult β-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The −113A>G HPFH mutation falls within the −115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the −113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the −113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the −113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.

The prevalence and molecular characterization of (δβ)0 -thalassemia and hereditary persistence of fetal hemoglobin in the Chinese Zhuang population.

ObjectiveTo reveal the prevalence and molecular characterization of (δβ)0‐thalassemia [(δβ)0‐thal] and hereditary persistence of fetal hemoglobin (HPFH) in the Chinese Zhuang population.MethodsA total of 105 subjects with fetal hemoglobin (Hb F) level ≥5% from 14 204 unrelated ones were selected for the study. Multiplex ligation dependent probe amplification was firstly used to analyze dosage changes of the β‐globin gene cluster for associated with (δβ)0‐thal and HPFH mutations. The gap polymerase chain reaction was then performed to identify the deletions using the respective flanking primers. Hematologic data were recorded and correlated with the molecular findings.ResultsTwenty‐one (0.15%) subjects were diagnosed with Chinese Gγ(Aγδβ)0‐thal. Nine (0.06%) were diagnosed with Southeast Asia HPFH (SEA‐HPFH) deletion. Seventy‐five (0.53%) cases remained uncharacterized. Three genotypes for Chinese Gγ(Aγδβ)0‐thal and SEA‐HPFH deletion were identified, respectively. The genotype‐phenotype relationships were discussed.ConclusionOur study for the first time demonstrated that (δβ)0 and HPFH were not rare events, and molecular characterized Gγ(Aγδβ)0‐thal and HFPH mutations in the Chinese Zhuang population. The findings in our study will be useful in genetic counseling and prenatal diagnostic service of β‐thalassemia in this populations.

An HPFH Mutation in the γ-Globin Gene Promoter of Humanized Cooley’s Anemia Mice Rescues Them from Perinatal Lethality and Renders Them Transfusion Independent

Cooley’s Anemia (CA), β-thalassemia major, is a genetic disease caused by an impairment in β-globin protein synthesis. The resulting excess in α-globin chains causes the premature destruction of erythroid cells (ineffective erythropoiesis), anemia, and if left untreated, death within the first years of life. Several mutations in the promoters of the fetal γ-globin genes have been identified which impair the silencing of the fetal genes in adulthood, a condition termed hereditary persistence of fetal hemoglobin (HPFH). The amount of fetal hemoglobin (Hb F) expressed and the distribution within the RBC population (pancellular vs heterocellular), varies widely with different HPFH mutations. It has also been observed that thalassemia patients who co-inherit HPFH mutations that express higher levels of Hb F have milder disease symptoms. The purpose of this study is to determine whether the incorporation of a non-deletional HPFH mutation into the promoter of the human g-globin gene in a humanized mouse model of CA can rescue the animals from their perinatal lethality. Heterozygous humanized HPFH -175 mice express pancellular, high-levels of Hb F into adulthood. Homozygous HPFH -175 CA mice are rescued from their perinatal lethality, surviving solely on 100% human Hb F and are transfusion independent for life. This result is significant because it demonstrates that introduction of an HPFH mutation into the γ-globin gene promoter by gene editing may be a viable therapeutic option for CA patients in the future. DisclosuresNo relevant conflicts of interest to declare.

Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

Generation of Non-Deletional Hereditary Persistence of Fetal Hemoglobin (HPFH) Beta-Yac Transgenic Mouse Models: -175 Black HPFH and -195 Brazilian HPFH

Fetal hemoglobin (HbF) is a major genetic modifier of the phenotypic heterogeneity in patients with the major β-globin disorders sickle cell disease (SCD) and β-thalassemia. Although the normal level of HbF postnatally is approximately 1% of total hemoglobin, some individuals have a condition known as hereditary persistence of fetal hemoglobin (HPFH), characterized by elevated synthesis of γ-globin in adulthood. HPFH is caused by small or large deletions in the β-globin locus (deletional HPFH), or point mutations in the Aγ-globin or Gγ-globin gene promoters (non-deletional HPFH). Pharmacological agents such as butyrate, decitabine, and hydroxyurea are effective in inducing HbF in vitro and in vivo. To date, hydroxyurea is the only drug approved for clinical use in sickle cell patients, although the efficacy level is variable between patients and the long-term effects of this drug remain uncertain. Therefore, current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin gene expression in adult life. Many studies have demonstrated the role of stage-specific transcription factors in β-like globin gene switching, indicating their potential as therapeutic targets in the treatment of β-hemoglobinopathies. In order to better understand the molecular pathways involved in the regulating γ-globin gene expression, we used β-YAC transgenic mice, produced with a 213 Kb β-globin locus yeast artificial chromosome, containing a 187 Kb human chromosomal insert encompassing the entire 82 Kb β-globin locus from 5'HS5 of the LCR to 3'HS1, approximately 20 Kb downstream from the β-globin gene. Four different transgenic mouse lines were included in this study: 1) wild β-YAC mice, with the normal sequence of the human β-globin locus; 2) mutant β-YAC mice with the Aγ-globin -117 G>A HPFH mutation 3) mutant β-YAC mice with the Aγ-globin -175 T>C HPFH mutation, and 4) mutant β-YAC mice with the Aγ-globin -195 T>C HPFH mutation. Adult -175 and -195 mutant β-YAC mice displayed an HPFH phenotype with an increased level of HbF. As measured by HPLC, -175 HPFH mice had the highest average level of γ-globin chains [16.4% γ/(γ+β)], followed by -195 HPFH mice (8.4%). Wild-type β-YAC control mice averaged 2.8% and -117 Greek HPFH β-YAC control mice displayed an average of 7.4%. Measurement of Aγ-globin mRNA by RNase protection analysis (RNAP) supported the HPLC data; γ/(γ+β) was 34%, 12.1%, 14.1% and less than 0.5% for -175 HPFH, -195 HPFH, -117 HPFH and wild-type β-YAC animals, respectively. Relative mRNA levels as determined by RT-qPCR were consistent with the RNAP results. Currently, we are examining our -175 and -195 HPFH mice for pancellular versus heterocellular distribution of HbF. To examine the molecular basis for the -175 and 195 HPFH phenotypes, fetal livers of these animals were collected on day E18 of gestation, after the fetal-to-adult β-like globin switch occurred, for chromatin immunoprecipitation (ChIP) analysis of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2 and LDB1. Previous unpublished DNA-protein array and ChIP data, comparing human primary erythroid cell cultures from normal donors and -195 HPFH individuals, showed a 6-fold enrichment of YY1 recruitment to the -195 region of the normal Aγ-globin promoter and a 5-fold enrichment of PAX1 recruitment to the HPFH mutant promoter, suggesting that YY1 may act as an A γ-globin gene repressor and PAX1 may be an activator when the -195 mutation is present. Preliminary ChIP experiments in β-YAC mice showed a similar pattern with YY1 enriched 2-fold in wild-type mice and PAX1 enriched 2-fold in -195 HPFH animals. Regarding -175 HPFH and wild-type β-YAC samples, we found occupancy enrichment of LMO2, TAL1 and LDB1 proteins (1.5-fold, 9-fold and 2.5-fold, respectively) in the -175 region of the Aγ-globin gene promoter in -175 HPFH β-YAC mice. Recently published studies in cell lines have shown that these three proteins form a complex with GATA-1 to mediate long-range interactions between the LCR and β-like globin genes. These mouse models provide additional tools for studying the regulation of γ-globin gene expression and may reveal new targets for selectively activating HbF. DisclosuresNo relevant conflicts of interest to declare.

Genome Editing Recreates Hereditary Persistence of Fetal Hemoglobin in Primary Human Erythroblasts

Manipulating the developmental switch from γ- to β-globin expression that occurs after birth has been intensively investigated as therapeutic strategy for sickle cell anemia and β-thalassemia. Rare individuals with a benign condition termed hereditary persistence of fetal hemoglobin (HPFH) exhibit an attenuated or absent γ-to-β switch, resulting in high levels of fetal hemoglobin (α2γ2) in all red blood cells (RBCs) throughout life. Moreover, individuals with HPFH and homozygosity for sickle cell disease (SCD) mutations exhibit few or no clinical manifestations of the latter. We used genome editing to induce a naturally occurring 13-nucleotide (-102 to -114) deletional HPFH mutation in the γ-globin (HBG1) gene promoter. Heterozygosity for this mutation is associated with HbF levels > 30% in adults. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to create small deletions around -102 to -114 in the γ-globin genes in peripheral blood CD34+ cells from healthy donors. We delivered guide RNA (gRNA) and Cas9 using lentiviruses, sorted transduced hematopoietic progenitors by FACS, and cultured them using a 3-phase erythroid differentiation protocol. Real time PCR showed that γ-globin mRNA increased more than 10-fold in Cas9/gRNA transduced cells compared to controls. HbF flow cytometry and high-performance liquid chromatography (HPLC) demonstrated that induced γ-globin chains were effectively incorporated into hemoglobin tetramers. HPLC revealed 1-3% HbF in negative controls and an increase to 15% in cells transduced with gRNA and Cas9. Expression of erythroid differentiation markers CD235 and CD71 were unaffected, suggesting that the γ-globin increase is not due to impaired erythroid maturation. Next generation sequencing demonstrated that a single gRNA created one predominant mutation that co-segregated with high HbF expression and represented over 50% of the sequencing coverage. Interestingly, this mutation is identical to the 13-nucleotide HPFH deletion. We also tested the gRNA mutation efficiency after transient expression of gRNA and Cas9 in human CD34+ cells by electroporation followed by analysis of single burst-forming unit-erythroid (BFU-E) colonies formed in methylcellulose. Genomic DNA analysis revealed that one gRNA targeted 50% of HBG1 alleles, and cells that received two overlapping gRNAs demonstrated 80% mutation frequency. Real-time PCR of mRNA from edited BFU-Es showed that mutations stimulated γ-globin mRNA expression to 19-55% total globin synthesis, whereas control colonies contained 1-5% γ-globin. Together, our data demonstrate that the CRISPR-Cas9 system can generate precisely the -102 to -114 HPFH mutation at high efficiency in primary human progenitor cells and thereby induce the expression of HbF to potentially therapeutic levels. This work provides proof of concept for targeted genome editing for γ-globin activation as a therapy for patients with β hemoglobinopathies. DisclosuresWeiss:Biogen: Research Funding; GlaxoSmithKline: Consultancy; Rubius: Membership on an entity's Board of Directors or advisory committees.

Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.

Evaluation of HPFH and δβ-thalassemia mutations in a Brazilian group with high Hb F levels

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Hereditary Persistence of Fetal Hemoglobin: Old, New and Future Mutations in the aγ-Globin Gene-Proximal Region

Hereditary persistence of fetal hemoglobin (HPFH) is a result of mutations that prevent the silencing of the g-globin genes during the adult stage of definitive erythropoiesis. Two types of HPFH are recognized, deletional HPFH and non-deletional HPFH. Mutations in the later class have been identified in the proximal promoters of the Ag- and Gg-globin genes. Individuals homozygous for sickle cell disease or certain b-thalassemia mutations, that have in addition a HPFH mutation, do not suffer the deleterious effects of these diseases. These subjects provide the natural evidence supporting the clinical effort to reactivate fetal hemoglobin as the major treatment for SCD and b-thalassemias. Thus, understanding the molecular mechanisms regulating the g-globin genes is essential for identification of points of therapeutic intervention. Although the number of point mutations causing HPFH has grown over the years, the biochemical mechanisms affected by these alterations remains elusive. In addition, it is unlikely that all potential mutations have been identified in humans. A complete catalog of all potential HPFH point mutations, coupled with knowledge of the transcriptional processes affected by them will be an invaluable step towards effectively treating these diseases. We recently identified a novel T>A HPFH mutation in a GATA site at position -566 of the Ag-globin promoter, the most distal in the promoter to date, that affects binding of a GATA-1-FOG-1-Mi2 repressor complex. Since this study utilized mutated human b-globin locus yeast artificial chromosome (b-YAC) transgenic mice, where a second copy of the Ag-globin gene was introduced near the locus control region, we produced b-YAC transgenic mice containing the -566 mutation at the normally located Ag-globin gene. These mice display a mild HPFH phenotype, an approximately 3% increase in g-globin gene expression, compared to wild-type b-YAC mice. Chromatin immunoprecipitation (ChIP) studies demonstrated that this mutation prevents GATA-1 binding when g-globin is repressed in post-conception day 18 (E18) fetal liver, whereas recruitment was observed in wild-type b-YAC transgenic samples from the same developmental stage. These data are consistent with the presence of a GATA-1-mediated repressor complex at this GATA site when g-globin is not expressed. GATA-1-mediated repression may be a general mechanism of g-globin silencing. To begin testing this hypothesis, we utilized previously generated Ag-globin -117 G>A Greek HPFH b-YAC transgenic mice, which show a 5–8% increase in g-globin synthesis in adult erythropoiesis. Published data suggested that this mutation affects nearby GATA-1 binding. Our ChIP data confirmed these results, however the GATA-1 multi-protein complex that is affected may differ from that recruited to the -566 GATA binding site. Finally, we have developed a cell-based selection that is being used to identify a comprehensive set of Ag-globin HPFH promoter mutations. Chemical inducer of dimerization (CID)-dependent Ag-globin promoter-eGFP b-YAC bone marrow cells were derived from transgenic mice and mutagenized with N-ethyl, N-nitrosourea (ENU). These cells are normally GFP−; treatment with g-globin-inducers or the presence of the -117 Greek HPFH mutation results in GFP+ cells. GFP+ cells were collected by FACS and individual cell clones expanded so that genomic DNA could be isolated. Promoter proximal regions were amplified by four PCR primer sets and subjected to heteroduplex analysis with the corresponding wild-type Ag-globin promoter PCR products as the control amplicons. Twenty three heteroduplexes have been detected among 158 mutant clones screened. Most are clustered in the proximal promoter. These data suggest that we have produced HPFH mutations, likely consisting of those known in human populations, as well as novel sites that affect repressor binding or enhance recruitment of transcriptional activators.

Hemoglobin variants, hematological parameters and β-globin gene cluster haplotypes in an isolated Amerindian group from the Orinoco River Delta

Background: Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics.Aim: The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits.Subjects and methods: Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, γ-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta.Results: Two βs genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. β-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%.Conclusions: The presence of the HPFH mutation was confirmed, and the new β-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988).

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the γ-globin gene in adult erythropoiesis

Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal gamma-globin gene results in reactivation of the gamma-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced beta locus YAC transgenic mice carrying an exact beta locus replicate of a deletional HPFH mutation, HPFH 2. Although the gamma-globin gene was expressed in the HPFH 2/beta locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of gamma-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the gamma-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the beta-YAC construct might be involved in gamma-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.

The Venezuelan Type of Nondeletional Hereditary Persistence of Fetal Hemoglobin Is Due C →T Mutation at Nucleotide -211 of the Aγ Globin Gene.

Hereditary Persistence of Fetal Hemoglobin (HPFH) is a clinically benign condition characterized by continuous synthesis of fetal hemoglobin (Hb F) in adult life without major related hematological changes. The nondeletional HPFH is characterized by hemoglobin F level between 5% and 30%, in heterozygotes state. This condition has been observed among Blacks, Italians, Greeks, English and Chinese. Two different forms are known, characterized by the type of chain involve, either Gγ or Aγ. Several point mutations in the promoter region of the Aγ globin gene have been identified in ndHPFH: −202, −198, −196, −195, −117, −114, deletion 4pb (−222 −225) and −158.The propositus is a 36 year old black female with a moderate increased level of Hb F, also her sister and her sibling had the same condition. She was discovered during studies due to the presence of splenomegaly and gastroesophageal reflux. The Hbs A, A2 and F were eluted and quantified by cation exchange high-performance liquid chromatography (HPLC-CE), using Beta Tal short program, Variant BioRad (Hercules, California, USA). DNA was isolated from peripheral blood leukocytes by a salting-out extraction procedure. The Aγ and Gγ promoters globins gene were amplified independently by PCR and the presence of the HPFH mutation was determined by denaturing gradient gel electrophoresis technique (DGGE) as previously described Gottardi et al., (1992). The Aγ promoter globin gene was sequenced in three members of the family using the BigDyeTM Terminator Cycler Sequencing Kit and the ABI PRISMTM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CQ, USA).Her hematological studies revealed Hb: 14.2 g/dl, Hcto: 41.1 %, MCV: 94.4 fl, MCHC: 34.5 fl, Hb A: 81.9 %, Hb A2: 2.1 % and Hb F: 6.3 %. The sibling hematological finding was Hb: 13.1 g/dl, Hcto: 41.8 %, MCV: 83.7 fl, MCHC: 31.3 fl, Hb A: 83.2 %, Hb A2: 3.2 % and Hb F: 5.1. The sister was Hb: 14.3 g/dl, Hcto: 46.4 %, MCV: 105.9 fl, MCHC: 30.8 fl, Hb A: 83.7 %, Hb A2: 3.3 % and Hb F: 2.7 %. No spleen enlargement was seen in the other two affected members of the family. The DGGE results in the Aγ promoter, suggested the presence of a non-deletion form of HPFH in heterozygotes state. The Aγ promoter region revealed the presence of a substitution C→T at position −211. This mutation does not modify any restriction site.To our knowledge the −211 (C→T) mutation in the Aγ promoter have not been reported previously. Since modulation of g-globin gene expression in adults could provide a therapeutic intervention in hemoglobinopathy, the study of the molecular mechanisms generating HPFH is medically relevant.