Evidence for the in vivo safety of insulated foamy viral vectors.

Retroviral vector-mediated stem cell gene therapy is a promising approach for the treatment of hematopoietic disorders. However, genotoxic side effects from integrated vector proviruses are a significant concern for the use of retroviral vectors in the clinic. Insulated foamy viral (FV) vectors are potentially safer retroviral vectors for hematopoietic stem cell gene therapy. We evaluated two newly identified human insulators, A1 and A2, for use in FV vectors. These insulators had moderate insulating capacity and higher titers than previously developed insulated FV vectors. The A1-insulated FV vector was chosen for comparison with the previously described 650cHS4-insulated FV vector in human cord blood CD34+ repopulating cells in an immunodeficient mouse model. To maximize the effects of the insulators on the safety of FV vectors, FV vectors containing a highly genotoxic spleen focus forming virus promoter were used to elicit differences in genotoxicity. In vivo, the A1-insulated FV vector showed an approximate 50% reduction in clonal dominance compared with either the 650cHS4-insulated or control FV vectors, although the transduction efficiency of the A1-insulated vector was higher. This data suggests that the A1-insulated FV vector is promising for future preclinical and clinical studies.

A capsid-modified adenovirus vector devoid of all viral genes: assessment of transduction and toxicity in human hematopoietic cells.

Inefficient gene transfer has limited the success of gene therapy in the hematopoietic system. Here we develop a novel chimeric adenovirus (Ad) vector containing Ad serotype 11 fiber-modified capsids and E1/E3 deleted viral genomes (Ad5/11) or genomes devoid of all viral genes (DeltaAd5/11). The capsid-modified vectors transduced human hematopoietic cells more efficiently than the unmodified Ad5-based vector. The absence of viral genes from the DeltaAd5/11 vector allowed for transduction without the associated toxicity seen with the first-generation E1/E3 deleted vector. Chimeric vectors were used for transient expression of the ecotropic retrovirus receptor (ecoR) in Mo7e cells (a CD34-positive, c-Kit-positive, growth-factor-dependent human cell line) as a model for human hematopoietic progenitor cells. Expression of ecoR conferred susceptibility to subsequent retroviral transduction. The DeltaAd5/11 vector used to express ecoR allowed for expansion of retrovirally transduced cells, whereas transduction with the first-generation Ad5/11 vector resulted in cytotoxicity and, over time, loss of cells expressing the retrovirus-vector-derived transgene.

Role of NF-Y in in vivo regulation of the gamma-globin gene.

The duplicated CCAAT box is required for gamma gene expression. We report here that the transcriptional factor NF-Y is recruited to the duplicated CCAAT box in vivo. A mutation of the duplicated CCAAT box that severely disrupts the NF-Y binding also reduces the accessibility level of the gamma gene promoter, affects the assembly of basal transcriptional machinery, and increases the recruitment of GATA-1 to the locus control region (LCR) and the proximal promoter and the recruitment of transcription cofactor CBP/p300 to the LCR. These findings suggest that recruitment of NF-Y to the duplicated CCAAT box plays a role in the chromatin opening of the gamma gene promoter as well as in the communication between the gamma gene promoter and the LCR.

Cloning and characterization of a potential transcriptional activator of human gamma-globin genes.

Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human gamma-globin and switch to human beta globin expression as time in culture advances. One explanation for the initially predominant expression of gamma-globin gene in these hybrids is the presence of trans-acting factors that activate gamma-globin gene transcription. We used differential display of hybrids before and after the gamma to beta switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential gamma gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only gamma-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to beta globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of gamma mRNA in HFE x MEL cell hybrids that undergo the gamma to beta switch, suggesting that this new gene encodes a protein that may function as gamma gene activator.

A chromatin insulator protects retrovirus vectors from chromosomal position effects.

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed in vitro and in vivo. To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken beta-globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin.

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human gamma and beta globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific gamma globin gene inducers are recognized by their ability to increase gamma-firefly luciferase (gamma(F)) gene activity significantly more than beta-renilla luciferase (beta(R)) gene activity, identified by an increased ratio of gamma-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase gamma-globin gene expression. It can therefore be used for the screening of chemical agents that may have gamma-globin gene inducibility.

FKLF-2: a novel Krüppel-like transcriptional factor that activates globin and other erythroid lineage genes.

FKLF-2, a novel Krüppel-type zinc finger protein, was cloned from murine yolk sac. The deduced polypeptide sequence of 289 amino acids has 3 contiguous zinc fingers at the near carboxyl-terminal end, an amino-terminal domain characterized by its high content of alanine and proline residues and a carboxyl-terminal domain rich in serine residues. By Northern blot hybridization, the human homologue of FKLF-2 is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in established cell lines with an erythroid phenotype, but it is inconsistently expressed in cell lines with myeloid or lymphoid phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is up-regulated after induction of mouse erythroleukemia cells. In luciferase assays, FKLF-2 activates predominantly the gamma, and to a lesser degree, the epsilon and beta globin gene promoters. The activation of gamma gene promoter does not depend on the presence of an HS2 enhancer. FKLF-2 activates the gamma promoter predominantly by interacting with the gamma CACCC box, and to a lesser degree through interaction with the TATA box or its surrounding DNA sequences. FKLF-2 also activated all the other erythroid specific promoters we tested (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase). These results suggest that in addition to globin, FKLF-2 may be involved in activation of transcription of a wide range of genes in the cells of the erythroid lineage.

Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector.

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region.

The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid lineage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remains unknown. To gain insights into its function, mouse HS5 was cloned and sequenced. Comparison of the HS5 sequences of mouse, human, and galago revealed two extensively conserved regions, designated HS5A and HS5B. DNase I hypersensitivity mapping revealed that two hypersensitive sites are located within the HS5A region (designated HS5A(major) and HS5A(minor)), and two are located within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, suggesting that these protein binding sites are implicated in the formation of HS5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific. HS5 was detected in murine brain but not in murine kidney or adult liver, suggesting that this site is not ubiquitous. The presence of GATA-1 and NF-E2 motifs (which are common features of the DNase I hypersensitive sites of the LCR) suggests that the HS5 is organized in a manner similar to that of the other HSs. Taken together, our results suggest that HS5 is an inherent component of the beta-globin locus control region.

Stem cell gene therapy for the beta-chain hemoglobinopathies. Problems and progress.

Virus vectors hold great promise for the stem cell gene therapy of beta-chain hemoglobinopathies. However, conventional vectors suffer from low gene transfer rates, low expression levels, and inconsistent or short-lived expression in vivo. In this review we summarize the current status of vector systems for the transduction of hematopoietic stem cells, including the development of novel vector systems and methods for selection of transduced stem cells in vivo. We also summarize efforts to achieve therapeutic expression levels of transferred globin genes with retrovirus vectors, including the manipulation of transcription cassettes, the use of globin gene enhancers, and advances in the use of chromatin insulators for improving the frequency of gene expression following hematopoietic stem cell transduction.

Analysis of gamma-globin expression cassettes in retrovirus vectors.

With the goal of optimizing retrovirus vectors for human gamma-globin, we studied the effect of several globin gene expression elements on vector titer, stability, and expression. We found that all combinations tested were genetically stable, but that vectors with therapeutic titers (0.5 to 2 x 10(6) colony-forming units/ml) could be achieved only by either partially or fully deleting the second intron of the Agamma-globin gene. Efficient transfer and high-level expression was achieved only when an optimized beta-globin promoter was linked to an Agamma-globin cassette containing an intact intron 1 and a 714-bp internal deletion of intron 2. When flanked by two copies of the HS-40 enhancer core from the alpha-globin locus, this cassette expressed gamma-globin mRNA at 46 +/- 19% per copy of mouse alpha-globin in the murine erythroleukemia cell line MEL585. Complete deletion of the first or second intron diminished expression to < or = 2.0%, and deletion of the HS-40 enhancer diminished expression to 7 +/- 8%. High-level, uniform expression of gamma-globin protein was confirmed in MEL585 clones (n = 12) transduced with the optimized vector. Efficient but variable expression of the optimized vector was also observed in erythroid progenitor colonies (n = 6) grown from transduced mouse bone marrow. Taken together, these studies demonstrate the role of intronic, promoter, and enhancer sequences on retrovirus vectors for human gamma-globin, and the development of an optimized vector capable of efficient expression in a murine erythroid cell line and primary cultures.

FKLF, a novel Krüppel-like factor that activates human embryonic and fetal beta-like globin genes.

A cDNA encoding a novel Krüppel-type zinc finger protein, FKLF, was cloned from fetal globin-expressing human fetal erythroid cells. The deduced polypeptide sequence composed of 512 amino acids revealed that, like Sp1 and EKLF, FKLF has three contiguous zinc fingers at the near carboxyl-terminal end. A long amino-terminal domain is characterized by the presence of two acidic and two proline-rich regions. Reverse transcription (RT)-PCR assays using various cell lines demonstrated that the FKLF mRNA is expressed predominantly in erythroid cells. FKLF message is detectable by RT-PCR in fetal liver but not in adult bone marrow cells. As predicted from its structural features, FKLF is a transcriptional activator. In luciferase assays FKLF activated the gamma- and epsilon-globin gene promoters, and, to a much lower degree, the beta-globin promoter. Studies of HS2-gamma gene reporter constructs carrying CACCC box deletions revealed that the CACCC box sequence of the gamma gene promoter mediates the activation of the gamma gene by FKLF. Other erythroid promoters (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase) containing CACCC elements or GC-rich potential Sp1-binding sites were activated minimally, if at all, by FKLF, indicating that FKLF is not a general activator of genes carrying the CACCC motifs. Transfection of K562 cells with FKLF cDNA enhanced the expression of the endogenous epsilon- and gamma-globin genes, suggesting an in vivo role of FKLF in fetal and embryonic globin gene expression. Our results indicate that the protein potentially encoded by the FKLF cDNA acts as a transcriptional activator of embryonic and fetal beta-like globin genes.

Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette.

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

Correct function of the locus control region may require passage through a nonerythroid cellular environment.

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Predominant expression of human Agamma--in contrast with beta-globin gene in MEL cells transfected with the construct muLCRAgamma psibeta deltabeta.

A cosmid construct muLCRAgamma psibeta deltabeta were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM-979 that expresses both murine embryonic and adult globins. Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct. Inducible expression of the Agamma- and beta-gene was observed during induced cell differentiation. However, the expression level of the Agamma-globin gene is much higher than that of the beta-globin gene in either uninduced or induced MEL transformants. No gamma to beta switching happened in the stable MEL transformants following a continuous culture. The much more effective enhance of the muLCR on the Agamma-globin gene than that on the beta-globin gene is resulted probably from the fact that the distance between the LCR and the beta-globin gene is much longer than that between the LCR and the Agamma-globin gene in the construct, in comparison with other constructs containing HS2 or muLCR linked to both of gamma- and beta-globin genes in different order. Two suggestions can be derived from these results: 1) A competition between the gamma- and beta-globin gene for interaction with the LCR may indeed present, but only an enough long distance difference between the LCR to the gamma- and to the beta-gene can effectively influence the competition; 2) Unlike transgenic mice, MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the beta-globin gene cluster with limited length.

Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin.

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice.

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.

Analysis of enhancer function of the HS-40 core sequence of the human alpha-globin cluster.

HS-40 is the major regulatory element of the human alpha-globin locus, located 40 kb upstream of the zeta-globin gene. To test for potential interactions between HS-40 and the beta- or the gamma-globin gene promoters in stable transfection assays, the HS-40 core sequence was cloned upstream of either the beta promoter or the gamma promoter driving the neomycin phosphotransferase gene and enhancer activity was measured using a colony assay. In K562 or in MEL cells, enhancer activity of HS-40 was higher than that of the individual core sequences of the DNase I hypersensitive sites (HS) of the beta-globin locus control region (LCR), and approximately 60% of the enhancer activity of a 2.5 kb microLCR, which contains the core elements of DNase I hypersensitive sites 1-4. In contrast to the synergistic interaction between the DNase I hypersensitive sites of beta locus LCR, combination of HS-40 with these DNase I hypersensitive sites failed to display cooperativity in K562 cells and inhibited enhancer function in MEL cells. Inhibition of enhancer function was also observed when two copies of the HS-40 were arranged tandemly. We conclude that the core element of HS-40 (i) is a powerful enhancer of gamma- and beta-globin gene expression, (ii) in contrast to other classical enhancers, acts best as a single copy, (iii) does not cooperate with the regulatory elements of the beta-globin locus control region.

Stem cell gene therapy, position effects and chromatin insulators.

Low efficiency of gene transfer is the main obstacle for a clinically effective gene therapy at the level of the pluripotent hematopoietic stem cell. Another important aspect of stem cell gene therapy, the actual expression of the transduced genes, has only been investigated adequately in very few studies, mainly for globin genes. Transcriptional silencing and position effects due to negative effects of surrounding chromatin on the expression of randomly integrated vector sequences may seriously jeopardize the success of current gene therapy strategies, even if transduction efficiency can be significantly improved. We propose the incorporation of chromatin insulators in the design of gene therapy vectors to overcome the problem of position effects. Chromatin insulators are protein-binding DNA elements that lack intrinsic promoter/enhancer activity but shelter genes from transcriptional influence of surrounding chromatin. The best characterized insulators are from Drosophila. We hypothesize that the important cellular function of chromatin organization is evolutionarily conserved and that human homologs to Drosophila insulator binding proteins such as the suppressor of Hairy-wing exist and can be cloned. Using these putative proteins, it should be possible to identify corresponding minimal binding sites with insulator activity. The design and incorporation of effective chromatin insulator sequences in the next generation of gene therapy vectors should lead to improved and more predictable expression of therapeutic transgenes and constitute an important step toward clinically effective gene therapy.

Human CD34+ cell EST database: single-pass sequencing of 402 clones from a directional cDNA library.

A directional cDNA library was constructed from human CD34+ cells, and single-pass DNA sequence analysis was used to sequence 402 randomly picked cDNA clones. Sequence homology searches against the GenBank/EMBL databases revealed that 144 clones (35.8%) represented new genes, whereas 39 clones (9.7%) matched the partial sequences of cDNA clones (expressed sequence tags [ESTs]) from cDNA libraries from other tissues or cells. One hundred three (25.6%) of the clones were highly homologous or identical to genes of known function. 18.6% of the clones were ribosomal and 6 % were mitochondrial sequences. Seventeen (4.2%) of the clones were coding for Alu or L1 repetitive sequences. Our results suggest that random single-pass sequencing of cDNA clones provides a rapid way to obtain a profile of genes expressed in CD34+ cells and to identify new and potentially interesting genes.