The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus.

The cHS4 chromatin insulator has been shown to improve the expression of integrating gene transfer vectors by reducing the impact of silencing chromosomal position effects. To better understand the underlying mechanisms of this protection, we investigated the influence of this element on the epigenetic modifications of a gammaretroviral reporter vector. In HT1080 cells, we found that a fourfold increase in the level of green fluorescent protein (GFP) reporter expression from the cHS4-insulated vector was correlated with a twofold increase in acetylation at lysines 9 and 14 of histone H3, but not with CpG methylation. In a mouse bone marrow transduction and transplantation model, we found that a 10-fold increase in the likelihood of GFP expression from the cHS4-insulated vector was correlated with an eightfold increase in histone H3 acetylation, as well as a fourfold decrease in CpG methylation. Histone hyperacetylation peaked at the cHS4 core, and in vivo diminished nearly threefold through the central portion of the vector. Taken together, these studies demonstrate that the cHS4 chromatin insulator reduces gammaretroviral vector silencing by modulating epigenetic modifications of integrated provirus, and identify a specific topological distribution of these modifications that may prove informative for future vector designs.

Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin.

The development of oncoretrovirus vectors for human gamma-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of gamma-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the beta-globin locus control region to enhance the expression of an Agamma-globin gene with a truncated -382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for Agamma-globin linked to various combinations of the HPFH2 enhancer, the alpha-globin HS40 enhancer, and several versions of the promoter from Agamma-globin or beta-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoter-autonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a -201 bp Agamma-globin gene promoter with the Greek HPFH -117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248+/-99% per copy of mouse alpha-globin (62% of total alpha-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for Agamma-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the Agamma-globin promoter in the context of a gene transfer vector.

The emergence of the microstatistical paradigm in managed care.

Although commentators have argued that managed care constitutes a paradigm shift in the American health care system, it is argued that the guiding organizational assumptions driving orthodox managed care arrangements (i.e., HMOs and capitation) have been drawn from a paradigm that is 300 years old: the business of insurance. As such, traditional managed care directives have taken a macrostatistical organizational viewpoint. However well a macrostatistical paradigmatic view serves certain constructs such as the business of insurance, it is far too coarse a method for organizing and reforming the badly fragmented production processes of health care. As a corrective complement, a new microstatistical paradigmatic view is coming of age whose methodological directives are based in the episode of care, and which will ultimately allow for a more highly refined pricing and care process system to emerge.

Self-directed health plans: Web-enabled alternatives to traditional managed care.

New approaches for organizing, purchasing, and financing health care services are rapidly emerging as viable alternatives to orthodox managed care. One of the more intriguing approaches, Self-Directed Health Plans (SDHPs), empowers consumers as the agents of change and decision making in the continuing quest for health system reform. Combined with a host of newly evolved Internet support utilities, SDHPs represent a highly advanced paradigm for health insurance. Most importantly, SDHPs promise to genuinely harness the power and dynamism of free-market solutions in ways that are financially and socially sustainable, and to pick up the innovation process where health maintenance organizations left off. Thus, with SDHPs surfaces new hope that the problems facing America's health care system are not intractable to private sector creativity.

Tolerance to solid organ transplants through transfer of MHC class II genes.

Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tg's) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tg's, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts.

In vivo selection using a cell-growth switch.

A major obstacle to stem-cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant fraction of stem cells. One way to circumvent this obstacle is to use selection. Vectors containing two linked genes serve as the basis for selection, with one gene encoding a selectable product and the other, a therapeutic protein. Applying selection in vivo has the potential to bring a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically modified cells to be inducibly amplified, thereby averting the risks associated with cytotoxic drugs. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy.

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.

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors.

Nonhuman primates are increasingly being used as models for pre-clinical assessment of retrovirus vector expression and function following stem and progenitor cell transduction. We compared the relative susceptibility of CD34+ marrow progenitors from four nonhuman primate species and humans to transduction with amphotropic pseudotyped retrovirus vectors containing the Neo gene. The rate of functional gene transfer was measured by colony formation under G418 selection. Marrow progenitors from pigtail macaques (Macaca nemestrina) were transduced at about twice the rate (19.1 +/- 4.3%) as those from rhesus (11.2 +/- 3.7%) and cynomolgus (7.6 +/- 1.9%) macaques, baboons (7.8 +/- 1.8%), and humans (9.6 +/- 1.7%). Semiquantitative RT/PCR analysis suggests this difference may be due to elevated expression of the amphotropic receptor Pit2 in pigtailed macaque CD34+ cells. Further, transduction rates increased an average 1.6 +/- 0.4-fold when the culture temperature was lowered to 33 degrees C, and 2.1 +/- 0.3-fold when the culture dishes were coated with the fibronectin fragment CH-296. The data presented here point to important differences among nonhuman primate models as well as transduction culture conditions, and suggest that pigtailed macaques may be particularly useful for assessing expression and function of therapeutic retrovirus vectors. Gene Therapy (2000) 7, 359-367.

Global theory and the nature of risk, Part 2. Towards a choice-based model of managed care.

Orthodox managed care depends on top-down, command and control techniques to squeeze efficiency out of the system. But for every unit of economic good this approach produces, two or three bad units come as result. The key to moving to an environment where value and efficiency become self-sustaining is to structurally recognize the medicoeconomic reality of medicine: the episode of care. The episode forms a natural unit of analysis that not only renders costs and outcomes information translucent and accessible, but it also forms the natural conduit through which premium dollars can find their optimal value. By bifurcating probability risk from technical risk and allocating them in the ex ante and ex post markets, respectively, health care insurers and providers return to their rightful economic roles, and to their appropriate fiduciary duties. And patients regain some semblance of reasonable sovereignty in managing their own medical affairs.

Practical implementation of globally priced episodes of care: lessons learned from Oxford's specialty management program.

Globally priced episodes of care afford payers a more efficient method of allocating risk and simultaneously accommodating the now permanent demand for choice and access at the point of service. Foreseeing the trend away from capitated delivery systems, in April of 1996. Oxford Health Plans began to invest considerable resources to implement global fees. When Oxford ran afoul of Wall Street and New York State insurance regulators (for reasons unrelated to the subject of this article), it was forced to abandon the new program. For the growing number of payers interested in pursuing similar contracting strategies, this article may serve as a useful source of information.

Global theory and the nature of risk, Part I. How orthodox managed care stifles innovation.

The growing awareness that managed care is rapidly unraveling has not only produced a good deal of alarm, but also a call for prognostications regarding the future. Unfortunately, old habits die hard, and wedded ideologies die even harder. Instead of paving the way for innovation, most managed care pundits refuse to read the tea leaves properly and acknowledge that the orthodox regime is irretrievably comatose. Not understanding the fundamental flaws inherent in the old model, many persevere with rehashed predictions that only echo the very non-starters that got us in the present jam in the first place. Managed care has so far focused its energies on integrating the wrong objects, insurance and care, with all the predictably bad effects. Part 2 of this article will explore what this means and introduce the global theory of managed care as an alternative vision. Global theory lays a new foundation based on a more sound microeconomic model of risk, bifurcated markets, global fees for integrated episodes of care, and most important of all, patient/physician sovereignty.

Enhancement of swine progenitor chimerism in mixed swine/human bone marrow cultures with swine cytokines.

OBJECTIVE: The induction of transplantation tolerance across xenogeneic barriers by bone marrow transplantation holds great promise, but engraftment of xenogeneic stem cells has been difficult to achieve. Part of this difficulty is due to species-specific differences in regulatory cytokines and elements of the stromal microenvironment, which we studied here. MATERIALS AND METHODS: We developed a system where fresh bone marrow cells from swine and human are cultured on human bone marrow stroma in order to study these limiting factors in a clinically relevant species combination. RESULTS: We report here the ability of recombinant swine interleukin (IL)-3 and c-kit ligand (KL) to specifically enhance swine hematopoietic chimerism in this system. In the absence of exogenous swine cytokines, there were about half as many swine progenitors as human progenitors at 1, 2, and 4 weeks of culture. When used alone, swine IL-3 led to a notable but transient increase in the relative ratio of swine progenitors, while addition of swine KL increased the ratio of swine progenitors only modestly and only at later time points. In contrast, when swine IL-3 and KL were added together, there was a two- to fourfold increase in the ratio of swine to human progenitors at all times tested. CONCLUSION: These data demonstrate that both swine IL-3 and KL are needed for prolonged enhancement of swine progenitor chimerism under these conditions, and suggest that the species specificity of either one or both of these cytokines may represent an important barrier to prolonged engraftment of swine bone marrow in humans.

Integrated delivery systems: non fait accompli.

This article explores the themes that advocates of integrated delivery systems (IDSs) hoped would allow them to favorably leverage the final stages of managed care evolution: revenue generation, cost control, and quality improvement. In reality, however, IDSs have been highly unsuccessful in growing market share, competing against other managed care forms, and integrating delivery systems--their raison d'être. Two key concepts in global theory, risk and episodic management, are introduced, and utilized to show how IDSs addressed these fundamental properties inappropriately. As a result, financial results and system performance have been dismal. A prognosis is given as well as suggestions for improvement through risk bifurcation, global fees, and integrated episodes of care.

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.

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

BACKGROUND: Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS: To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS: Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION: To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

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.

Transfer of porcine MHC DRalpha into IEalpha-deficient murine bone marrow results in reduced IE-restricted Vbeta usage.

BACKGROUND: Allogeneic bone marrow transplantation has proven effective for inducing specific tolerance to subsequent solid organ allografts, although the clinical applicability of this approach is limited by the morbidity and mortality associated with this procedure. As an alternative, we are investigating the transfer of allogeneic MHC class II genes into recipient bone marrow cells (BMC), using the miniature swine as a model. METHODS: To understand the mechanism of tolerance induction achieved through class II gene transfer, BMC from C57BL/10 mice, which lack expression of the MHC class II DRalpha equivalent (H-2 IEalpha), were transduced with a retrovirus vector for swine DRalpha. RESULTS: Expression of the DRA-vector in bone marrow-derived cells was demonstrated by Northern analysis of colonies grown in vitro from transduced myeloid progenitors. Taking advantage of the fact that the introduced DRalpha chain was able to form heterodimers with endogenous IEbeta, surface expression of the transgene was demonstrated on splenocytes harvested 1, 17, and 28 weeks after bone marrow transplantation. Transgene expression was confirmed by reverse transcriptase-polymerase chain reaction in the thymus of those animals killed at weeks 17 and 28. Finally, the effects of bone marrow transduction on central tolerance induction was demonstrated by the progressive decrease of IE-reactive T-cell clones bearing Vbeta5 and Vbeta11 T cell receptors in the peripheral blood cells of engineered recipients. CONCLUSIONS: Our results support the notion that transplantation tolerance, induced by class II gene transfer into syngeneic BMC, results in part from durable deletional unresponsiveness of graft-specific alloreactive T cells.

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.

Targeted expansion of genetically modified bone marrow cells.

The ability to specifically target a mitogenic signal to a population of genetically modified primary cells would have potential applications both for gene and cell therapy. Toward this end, a gene encoding a fusion protein containing the FK506-binding protein FKBP12, fused to the intracellular portion of the receptor for thrombopoietin (mpl), was introduced into primary murine bone marrow cells. Dimerization of this fusion protein through the addition of a dimeric form of the drug FK506, called FK1012, resulted in a marked proliferative expansion of marrow cells that was restricted to the genetically modified population. FK1012's proliferative effect was sustained and reversible. An apparent preference for differentiation along the megakaryocytic lineage was observed. This approach allows for the specific delivery of a mitogenic signal to a population of genetically modified primary cells and may have applications for studies in hematopoiesis and receptor biology, and for gene and cell therapy.