Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros^mut248 mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.     

The clinical potential of stem cells

Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool.     

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O⁶-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders.     

Porphyrias: Animal models and prospects for cellular and gene therapy

The rapid progress in the development of molecular technology has resulted in the identification of most of the genes of the heme biosynthesis pathway. Important problems in the pathogenesis and treatment of porphyrias now seem likely to be solved by the possibility of creating animal models and by the transfer of normal genes or cDNAs to target cells. Animal models of porphyrias naturally occur for erythropoietic protoporphyria and congenital erythropoietic porphyria, and different murine models have been or are being created for erythropoietic and hepatic porphyrias. The PBGD knock-out mouse will be useful for the understanding of nervous system dysfunction in acute porphyrias. Murine models of erythropoietic porphyrias are being used for bone-marrow transplantation experiments to study the features of erythropoietic and hepatic abnormalities. Gene transfer experiments have been startedin vitro to look at the feasibility of somatic gene therapy in erythropoietic porphyrias. In particular, we have documented sufficient gene transfer rate and metabolic correction in different CEP disease cells to indicate that this porphyria is a good candidate for treatment by gene therapy in hematopoietic stem cells. With the rapid advancement of methods that may allow more precise and/or efficient gene targeting, gene therapy will become a new therapeutic option for porphyrias.     

Successful gene therapy of mice with congenital erythropoietic porphyria

Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut 248) mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.     

Technology for gene transfer into hematopoietic stem cells (Part 2)]

A hematopoietic stem cell is considered to be one of the ideal targets for gene therapy, and there is expectation that gene therapy will be established based on the technology of hematopoietic stem cell transplantation. However, in recent clinical trials of stem cell gene therapy for monogenic diseases, significant clinical improvement has not been reported. One of the main obstacles is the low efficiency of gene transfer into hematopoietic stem cells. Many investigators have been trying to improve the transduction efficiency to the clinically applicable level. Another approach to solve this problem is to develop the method for selective expansion of transduced hematopoietic stem cells in vivo. We are currently developing novel regulatory genes (selective amplifier genes) for stem cell gene therapy.     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.     

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.     

RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.     

Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1

Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.     

Human Immunodeficiency Virus Type 1-Induced Hematopoietic Inhibition Is Independent of Productive Infection of Progenitor Cells In Vivo

Human immunodeficiency virus (HIV)-infected individuals exhibit a variety of hematopoietic dysfunctions. The SCID-hu mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues) can be used to model the loss of human hematopoietic precursor cell function following HIV infection and has a distinct advantage in that data can be obtained in the absence of confounding factors often seen in infected humans. In this study, we establish that HIV type 1 (HIV-1) bearing a reporter gene inserted into the viral vpr gene is highly aggressive in depleting human myeloid and erythroid colony-forming precursor activity in vivo. Human CD34(+) progenitor cells can be efficiently recovered from infected implants yet do not express the viral reporter gene, despite severe functional defects. Our results indicate that HIV-1 infection alone leads to hematopoietic inhibition in vivo; however, this effect is due to indirect mechanisms rather than to direct infection of CD34(+) cells in vivo.     

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.     

Gene therapy using anticancer drug-resistance genes

Myelosuppression is a major dose-limiting factor in cancer chemotherapy. Introduction of drug-resistance genes into bone marrow cells of cancer patients has been proposed to overcome this limitation. In theory, any gene whose expression protects cells against the toxic effects of chemotherapy should be useful in vivo for this purpose. Among such genes, human multidrug-resistance gene (MDR1) has been studied most extensively for this purpose, and clinical trials of drug-resistance gene therapy have been started in the US for cancer patients who undergo high-dose chemotherapy with autologous hematopoietic stem cell transplantation. In Japan, our clinical protocol of MDR1 gene therapy "A clinical study of drug-resistance gene therapy to improve the efficacy and safety of chemotherapy against breast cancer" has been submitted to the government. To improve the efficacy and safety of this drug-resistance gene therapy, we have constructed a series of MDR1-bicistronic retrovirus vectors using a retrovirus backbone of Harvey murine sarcoma virus and internal ribosome entry site (IRES) from picornavirus to co-express a second gene with the MDR1 gene. MDR1-MGMT bicistronic vectors can be used to protect bone marrow cells of cancer patients from combination chemotherapy with MDR1-related anticancer agents and nitrosoureas. In addition, MDR1-bicistronic retrovirus vectors can be designed to use the MDR1 gene as an in vivo selectable marker to enrich the transduced cells which express therapeutic genes, if disease is curable by the expression of a single-peptide gene in any types of bone marrow cells or peripheral blood cells.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.     

全身放疗在造血干细胞移植中的研究进展

全身照射疗法(TBI)是一种姑息治疗,该方法已经成功地应用在慢性淋巴细胞白血病或滤泡性淋巴瘤等无干细胞支持的放射敏感的疾病中。目前,在血液系统恶性疾病中造血干细胞移植是较为有效的治疗手段之一,其中全身放射治疗与大剂量化疗是造血干细胞移植疗法的经典预处理方案。TBI方法主要应用在造血移植环境中,具有较强的周期非特异性抗肿瘤效应和免疫抑制效能。TBI给予干细胞移植病人超过正常骨髓的辐射耐受量,通过重建病人的造血和免疫来达到治疗目的。     

利用ES细胞建立可诱导的白血病模型

胚胎干细胞（embryonic stem cells,ESCs）是从囊胚的内细胞团分离出来的多潜能干细胞,具有多向分化的能力。将外源基因导入ES细胞建立转基因动物,对于研究外源基因的功能和调控具有一定的价值。载有外源性基因的病毒在感染ES细胞后,可通过囊胚注射获得具有胚系遗传的该转基因动物,并且这一外源基因可以稳定遗传和表达。该研究主要是利用携带hPML-RARα基因的慢病毒感染小鼠ES细胞系（R1）,获得携带该基因的ES细胞,感染后的ES细胞核型正常。在此基础上,将感染后的ES细胞经囊胚注射,获得了携带有hPML-RARα基因的3只嵌合小鼠,其中,有1只具有遗传特性。对嵌合体小鼠与C57杂交的后代给予强力霉素（doxycycline）处理,3天以后骨髓细胞hPML-RARα基因开始表达,这证明了在小鼠体内该外源基因表达的可诱导性。以上证实,已经成功利用ES细胞建立了可诱导的白血病转基因小鼠模型。     

Recent advances in gene therapy and future prospects

Gene therapy, which is treatment of diseases by introducing normal genes into the body, is becoming feasible as the result of advances in genetic engineering. The hematopoietic stem cells have been considered as the appropriate target for gene transfer in many genetic diseases for which allogeneic bone marrow transplantation has been employed successfully. However, there are still many problems to be solved. In particular, expression from retrovirally transduced genes in bone marrow cells has been transient and unstable. On the other hand, an alternative approach to somatic cell gene therapy using nonhematopoietic cells, including skin fibroblasts, endothelial cells, keratinocytes, and lymphocytes, has been shown to possess several advantages. This kind of approach is usually applied to supplementation therapy in not only hereditary disorders but also various acquired diseases, such as cancer or infectious diseases. Recently, clinical application of gene transfer into lymphocytes to treat cancer and immunodeficiency have been approved at NIH (USA). The trial could represent the start of a new era in molecular medicine.     

Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency

Background

Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency.

Methodology/Principal Findings

We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure.

Conclusions/Significance

Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.