Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

A gene conversion located 5' to the A gamma gene in linkage disequilibrium with the Bantu haplotype in sickle cell anemia.

Cloning and sequencing of the gamma-globin gene of a sickle cell anemia patient homozygous for the Bantu haplotype has revealed a gene conversion that involves the replacement of an A gamma sequence by a G gamma sequence in the promoter area of the A gamma gene. This event is similar to another gene conversion believed to be responsible for the very high homology between gamma-globin genes, suggesting that the promoter area of these genes is prone to this type of genetic rearrangement. Further analysis demonstrated that the chromosome bearing this gene conversion has a very high frequency among Bantu chromosomes and a very low or nil frequency in other haplotypes linked to the beta s gene. No correlation was found between the G gamma/A gamma ratio and the presence of the gene conversion among Bantu haplotype patients, thus excluding a portion of the gamma gene sequence in the determination of this ratio.

Enhancer-dependent transcriptional oscillations in mouse erythroleukemia cells.

By using recombinase-mediated cassette exchange, a method that allows integration of single copies of different constructs at the same predetermined chromosomal location, several expression cassettes have been integrated at a randomly chosen locus in the genome of mouse erythroleukemia cells. The cassettes studied contain the human beta-globin promoter fused to lacZ coding sequences either alone or linked to DNase I-hypersensitive site HS2, HS3, or HS234 (a large locus control region fragment containing HS2, HS3, and HS4) of the human beta-globin locus control region. Analysis of expression of these cassettes revealed mosaic expression patterns reminiscent of, but clearly different from, position effect variegation. Further investigations demonstrated that these mosaic expression patterns are caused by dynamic activation and inactivation of the transcription unit, resulting in oscillations of expression. These oscillations occur once in every few cell cycles at a rate specific for the enhancer present at the locus. DNase I sensitivity studies revealed that the chromatin is accessible and that DNase-hypersensitive sites were present whether or not the transcription unit is active, suggesting that the oscillations occur between transcriptionally competent and transcriptionally active chromatin conformations, rather than between open and closed chromatin conformations. Treatment of oscillating cells with trichostatin A eliminates the oscillations only after the cells have passed through late G1 or early S, suggesting that these oscillations might be caused by changes in histone acetylation patterns.

Position effects are influenced by the orientation of a transgene with respect to flanking chromatin.

We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement.

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.

The chicken beta-globin 5'HS4 boundary element blocks enhancer-mediated suppression of silencing.

A constitutive DNase I-hypersensitive site 5' of the chicken beta-globin locus, termed 5'HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophila cells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophila cells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated beta-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.

Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange.

Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.

Embryonic stem cells release potentially novel hematopoietic factors.

We have observed that a severe decrease in the number of erythroid progenitor cells follows the long-term culture (LTC) of human primitive stem cells on an established mouse fibroblast feeder layer. This suggests that one, or several, factors necessary to support erythroid differentiation might be missing in these culture conditions. To improve the erythroid differentiation and stem cell output of LTCs, we have examined the hypothesis that the factors that regulate pluripotent stem cell proliferation and erythroid commitment can be found in media conditioned by embryonic stem (ES) cells. We have found that media conditioned by undifferentiated and differentiating ES cells can affect differentiation patterns in both short-term culture and LTC assays. Medium conditioned by undifferentiated ES cells increases the numbers of estimated LTC-initiating cells (est.LTC-IC) and potentiates granulocytic differentiation. In contrast, medium conditioned by ES cells undergoing differentiation increases the number of est.LTC-IC and is a powerful promoter of erythroid differentiation. In the presence of this medium, the number of erythroid progenitors generated after 5 weeks of LTC was increased up to 100-fold as compared with controls, and the number of est.LTC-IC was amplified up to 140-fold. These results offer a new approach for the identification of factors implicated in stem cell proliferation, self-renewal and commitment. In addition, the improved LTC conditions for the expansion of stem cells without reduction of their in vitro differentiation potential should be useful for a wide range of applications.

A 6-bp deletion 5' to the G gamma globin gene in beta S chromosomes bearing the Bantu haplotype.

Sequencing of the upstream region of a human G gamma gene linked to the Bantu haplotype revealed a 6-bp deletion between site -400 and -395. Further analysis revealed that this mutation is present in 37% of the sickle cell anemia patients bearing the Bantu haplotype and is absent in the other haplotypes linked to the beta S gene, as well as in most chromosomes bearing the beta A-globin gene. The most parsimonious interpretation of the data is that the deletion is a very recent event which occurred in the subset of Bantu chromosomes already bearing a gene conversion of the A gamma gene by the G gamma gene. Its presence in black beta S chromosomes is most probably the consequence of a crossing-over between a Bantu beta S chromosome (with deletion and gene conversion) and a beta A chromosome.

[Analysis of the recombination zone in hereditary persistence of fetal hemoglobin with fetal gene rearrangement of the G gamma-G gamma-A gamma type]. / Analyse de la zone de recombinaison au cours d'une persistance héréditaire de l'hémoglobine foetale avec réarrangement des gènes foetaux de type G gamma-G gamma-A gamma.

An increased synthesis of fetal hemoglobin in adult life is a common feature of the genetically determined severe disorders like beta thalassemia and sickle cell anemia. A continued synthesis of fetal hemoglobin in adults is also characteristic of clinical or subclinical syndromes like respectively delta beta thalassemia or hereditary persistence of fetal hemoglobin (HPFH). These disorders are highly heterogeneous with respect to their molecular defects as well as to the composition of Hb F. We report here a novel case of hereditary persistence of fetal hemoglobin in heterozygous state discovered by chance, in a young perfectly healthy french man. The gamma chain of his fetal hemoglobin was almost entirely composed of G gamma chains. Molecular analysis of the DNA revealed the existence of triplicated gamma genes on one chromosome with the genotype arrangement of G gamma-G gamma-A gamma. A polymorphic Xmn I restriction site (at position -158 5' to the cap site) was present in 5' of both of these G gamma genes. The presence of this site in front of G gamma gene had previously been shown to be associated both with high G gamma phenotype constitutively and also with high fetal hemoglobin level only in case of anemic stress. In the absence of any anemic stress in this individual, the constitutive increase of both fetal hemoglobin and G gamma chains could be due to the presence of a chromosome with triplicated arrangement of gamma genes. The classical triplication (G gamma-A gamma-G gamma-A gamma) does not result in HPFH phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York.

Hemophilia B is an X-linked recessive bleeding disorder resulting from a deficiency of the coagulation factor IX (FIX) protein activity, a vitamin K-dependent serine protease active in both the intrinsic and extrinsic coagulation systems. DNA analyses of the factor IX gene in two unrelated patients with severe hemophilia B, with a IX coagulant activity less than 1% and undetectable FIX antigen, detected the loss of the second TaqI site in exon h (VIII) in both individuals. Polymerase chain reaction (PCR) amplification of 576 base pairs of exon h (VIII) with cloning and dideoxy sequencing of cloned DNA from one hemophiliac revealed a single C----T transition in codon 338 that changes an arginine residue codon CGA to a nonsense codon TGA. Allele-specific oligonucleotide probe hybridization with a mutant (C----T) and a wild-type allele confirmed the same mutation in amplified genomic DNA of the second hemophilia patient. The C----T transition represents another example of mutation at a CpG dinucleotide. DNA polymorphism analysis of the FIX gene in both individuals revealed each to be on a separate FIX haplotype; therefore, predicting each to be a separate mutation event.

Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange.

Efficient integration of transgenes at preselected chromosomal locations was achieved in mammalian cells by recombinase-mediated-cassette-exchange (RMCE), a novel procedure that makes use of the CRE recombinase together with Lox sites bearing different spacer regions. We have applied RMCE to the study of the human beta-globin gene Locus Control Region by integrating at the same genetic locus in MEL cells, a LacZ gene driven by the human beta-globin promoter linked to HS2 and HS3 alone or in combination with HS4. Expression studies at the cell population level and in individual cells before and after induction of differentiation with hemin or DMSO show that the presence of these enhancers is associated with variegated patterns of expression. We were able to show that the LCR fragments tested act by controlling both the probability of expression and the rate of transcription of the linked beta-globin promoter. Both of these factors were also dependent on the state of differentiation of the MELc and on the presence of a second transcription unit located in cis. The ability to manipulate by RMCE constructs integrated into chromosomes should help in the creation of complex, rationally designed, artificial genetic loci.

Polymerase chain reaction amplification applied to the determination of beta-like globin gene cluster haplotypes.

We report here on the application of the polymerase chain reaction (PCR) technique for the determination of the beta-like gene cluster haplotypes. Seven fragments containing each one of the following polymorphic sites--Xmnl 5' to the G gamma gene, HindIII in the IVSII of G gamma and A gamma gene, HincII 3' and inside the gamma gene, Hinfl 5' of the beta gene, and HpaI 3' of the beta gene--are amplified using the PCR technique. Each amplified fragment is subsequently digested with the appropriate enzyme, analyzed by electrophoresis on agarose gel containing ethidium bromide, and visualized under ultraviolet light. This technique has the advantages of rapidity, safety, and cost-effectiveness.

The enhancer-like sequence 3' to the A gamma gene is polymorphic in human populations.

Cloning and sequencing of the enhancer 3' of the A gamma globin gene of a particularly low G gamma and HbF sickle cell anemia (SCA) patient unexpectedly revealed three base changes (T----C, C----A, and A----G at sites +2285, +2460, and +2676) previously associated with the Seattle-type HPFH, thus leading the authors to suspect that the three mutations were polymorphic. The determination of the incidence of the mutations among various ethnic groups allowed the authors to conclude that this is a widely spread polymorphism, thus excluding any role of these base changes in the determination of the hereditary persistence of fetal hemoglobin (HPFH) phenotype. The origin of these three mutations is not clear because they appear linked, and the same bases (C, A, G) are found in homologous position in the 3' of the normal G gamma gene. As C, A, G at positions +2285, +2460, and +2676 are found with a 100% frequency in African SS patients and presumably among normal Africans (to explain the extremely high frequency among normal American blacks), it is likely that this was the sequence preceding the division of races. The presence of T, C, and A at the same positions apparently occurred after the divergence between blacks and the other races, that is, within the last 1 million years.

Nucleotide variations in the 3' A gamma enhancer region are linked to beta-gene cluster haplotypes and are unrelated to fetal hemoglobin expression.

Molecular cloning and sequence analysis of a nondeletion form of Sicilian beta o hereditary persistence of fetal hemoglobinemia (HPFH) (mutation in IVS2 nt1 position) homozygous for haplotype III revealed the presence of four sequence variations: C----T at -158 5' to G gamma, T----C at +2285, C----A at +2476, and A----G at +2676, all 3' to A gamma. The latter three variations in the putative A gamma enhancer are identical to those observed in the case of Seattle HPFH. However, a severe beta o-thalassemia case from Algeria (mutation in IVS1 nt1 position), also homozygous for haplotype III, revealed the same nucleotide variation, albeit an inefficient HbF production. We conclude that the variations in the A gamma enhancer element do not play a role in the regulation of HbF production. To assess both the linkage of these sites with the beta-cluster haplotype and the extent of the polymorphism, we examined several black and Mediterranean chromosomes, by PCR amplification followed by both EspI digestion and oligonucleotide hybridization. Our data indicate that these sequence variations in the enhancer element are absent in Mediterranean haplotypes I, V, and VII but are consistently associated with Mediterranean haplotypes II, III, and IX, as well as with the black beta c-associated haplotype. The common feature of all the latter haplotypes is the presence of a polymorphic PvuII site between A gamma and psi beta, which is thus in linkage disequilibrium with the variations in the A gamma enhancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of H1e histone linker-specific antibodies by means of synthetic peptides.

A large body of data suggests that the linker histones family (H1) affects gene expression. Investigation of the linker histones role is then of a major interest in cell cycle studies with implications in gene therapy. Indeed, it has been shown that in most tissues a switch of histone subtypes occurs when the cells cease to divide. To investigate linker histone role in gene or transgene expression, an antibody against subtypes of H1 would be useful for immunoprecipitation experiments and further assays measuring H1subtypes-DNA interactions in living cells. In order to produce an antibody against the H1e subtype of linker histones, two synthetic peptides derived from two regions of the H1e mouse histone protein were examined for their potential, [as keyhole limpet hemocyanin (KLH) conjugates] to elicit polyclonal anti-H1e antibodies in New Zealand white rabbits. Selection of the peptide sequences was based on amino acid differences within the different classes of histones and between mice and rabbit histones as well. The evaluation of their potential immunogenic properties was based on examination of peptide hydropathy using predicting algorithms. Immunoglobulins (IgG) obtained from immunized and nonimmunized rabbits were tested using enzyme-linked immunosorbent assay (ELISA) procedures, Western immunoblot, and immunofluorescence experiments. Results showed that the selected synthetic peptides gave rise to a high-titer polyclonal antibody able to recognize the H1e histone under various conditions. This polyclonal antibody did not cross-react with other histones. To our knowledge, this is the first antibody produced against the mouse H1e linker histone.

Non-erythroid genes inserted on either side of human HS-40 impair the activation of its natural alpha -globin gene targets without being themselves preferentially activated.

The human alpha-globin gene complex includes three functional globin genes (5'-zeta2-alpha2-alpha1-3') regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human alpha-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged alpha-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human alpha-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted alpha-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation.

The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients.

We have previously determined that in African sickle cell anemia (SS) patients three different beta-like globin gene cluster haplotypes are associated with different percent G gamma (one of the two types of non-alpha chains comprising hemoglobin F [HbF]), mean percent HbF, and percent dense cells. We report now that in adult New York SS patients, the presence of at least one chromosome with the Senegal haplotype is associated with higher Hb levels (1.2 g/dL higher) than is found for any other non-Senegal haplotype (P less than .004). The percent reticulocytes and the serum bilirubin levels were lower in these patients. When the effect of alpha-gene number was analyzed by examining a sample of SS patients with concomitant alpha-thalassemia, the same results were obtained. Because the HbF level is significantly higher among the Senegal haplotype carriers in this sample, the inhibitory effect on sickling of this Hb variant may be one of the reasons for the haplotype effect. We conclude that the Senegal beta-like globin gene cluster haplotype is associated with an amelioration of the hemolytic anemia that characterizes sickle cell disease.

An alanine-to-threonine substitution in protein 4.2 cDNA is associated with a Japanese form of hereditary hemolytic anemia (protein 4.2NIPPON).

Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2-deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele-specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2-deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.