Gene therapy of primary T cell immunodeficiencies

Gene therapy of severe combined immunodeficiencies has been proven to be effective to provide sustained correction of the T cell immunodeficiencies. This has been achieved for 2 forms of SCID, i.e SCID-X1 (γc deficiency) and adenosine deaminase deficiency. Occurrence of gene toxicity generated by integration of first generation retroviral vectors, as observed in the SCID-X1 trials has led to replace these vectors by self inactivated (SIN) retro(or lenti) viruses that may provide equivalent efficacy with a better safety profile. Results of ongoing clinical studies in SCID as well as in other primary immunodeficiencies, such as the Wiskott Aldrich syndrome, will be thus very informative.     

Cell therapy for inherited diseases of the hematopoietic system

Cell therapy was born in 1968 with the first allogeneic transplantation of hematopoietic stem cells for two immune deficiency disorders: the Wiskott-Aldrich syndrome and the Severe Combined ImmunoDeficiency (SCID). From this pioneering experience, thousands of patients affected with inherited or acquired diseases of the hematopoietic system have benefited from this therapeutic approach. Unfortunately, immunologic obstacles, represented by the compatibility in the major histocompatibility HLA system, still dictate today important limitations for a larger therapeutic utilization of hematopoietic stem cells (HSC). In this review, we have summarized the difficulties and the scientific advances leading us to improve the clinical results; the therapeutic research's track for primary immunodeficiencies is also discussed.     

Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg ^-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.     

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.     

Gene therapy of severe combined immunodeficiencies

The concept that the outcome of a devastating disease can be modified by inserting a transgene into abnormal cells is appealing. However, the gene-transfer technologies that are available at present have limited the success of gene therapy so far. Nevertheless, severe combined immunodeficiencies are a useful model, because gene transfer can confer a selective advantage to transduced cells. In this way, a proof of concept for gene therapy has been provided.     

造血干细胞生物学——研究与展望

造血干细胞（hematopoietic stem cell,HSC）是目前研究方法最为多样、研究技术手段最为成熟的一类组织干细胞,并且已经被成功运用于临床上对白血病以及先天性免疫缺陷等疾病的治疗。近年来,通过对一系列“转基因”与“基因敲除”小鼠模型的分析,人们对造血干细胞在胚胎早期发育过程中的发生与起源、造血干细胞“自我更新”与“定向分化”的调节机制、骨髓中造血干细胞的微环境（niche）对造血干细胞功能维持的调控,以及造血干细胞与白血病干细胞之间的相互关系等诸多方面都取得了很大的进展。如何实现造血干细胞的体外长期培养与扩增,实现胚胎干细胞（embryonic stem cell,ESC）或诱导多能干细胞（induced pluripotent stem cell,iPS细胞）向造血干细胞进行有效的定向分化,以及探索造血干细胞在病理状态（如癌症、贫血、衰老等）或应激状态下（如炎症与感染、组织损伤、代谢异常等）的功能变化,都将会是今后造血干细胞研究的重要方向。     

Abnormalities in DNA rearrangements of immunoglobulin gene loci in precursor B cells derived from X-linked agammaglobulinemia patient and a severe combined immunodeficiency patient

In an attempt to characterize the genes that cause immunodeficiencies such as X-linked agammaglobulinemia (XLA) and severe combined immunodeficiency (SCID) we established precursor B-cell lines by transforming the patients' bone marrow cells with Epstein-Barr viruses. DNA rearrangements of immunoglobulin J_H gene loci were observed on both chromosomes in pre-B cells derived from an XLA patient. We cloned and characterized both rearranged bands from one cell line. Both of the rearrangements occurred between D _H and J _H gene loci without the V_H DH structure. On the other hand, JH gene loci retained the germline configuration on both chromosomes in almost all the transformants derived from a SCID patient that had been determined according to their surface markers, to be in an early precursor B-cell stage. The implications of the observations are discussed.     

Hematopoietic stem cell expansion and gene therapy

Hematopoietic stem cell (HSC) gene therapy remains a highly attractive treatment option for many disorders, including hematologic conditions, immunodeficiencies including human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), and other genetic disorders such as lysosomal storage diseases. In this review, we discuss the successes, side-effects and limitations of current gene therapy protocols. In addition, we describe the opportunities presented by implementing ex vivo expansion of gene-modified HSC, as well as summarize the most promising ex vivo expansion techniques currently available. We conclude by discussing how some of the current limitations of HSC gene therapy could be overcome by combining novel HSC expansion strategies with gene therapy.     

Characterization of hematopoietic stem cells from the canine yolk sac

The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 10⁴ total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.     

Globin gene transfer for the treatment of severe hemoglobinopathies: a paradigm for stem cell-based gene therapy

The prospect of treating blood disorders with genetically modified stem cells is highly promising. This therapeutic approach, however, raises a number of fundamental biological questions, spanning several research fields. Further investigation is required to better understand how to isolate and efficiently transduce hematopoietic stem cells (HSCs), while preserving optimal homing and self-renewing properties; how to design safe vectors permitting controlled expression of the transgene products; and how to promote host repopulation by engrafted HSCs. This article addresses basic issues in stem cell-based gene therapy from the perspective of regulating transgene expression, taking globin gene transfer for the treatment of severe hemoglobinopathies as a paradigm.     

Expression of tumour‐suppressing chemokine BRAK/CXCL14 reduces cell migration rate of HSC‐3 tongue carcinoma cells and stimulates attachment to collagen and formation of elongated focal adhesions in vitro

BRAK/CXCL14 (breast‐ and kidney‐expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC‐3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock‐vector‐introduced (HSC‐3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high‐level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK‐dependent tumour suppression, we compared characteristics between HSC‐3 BRAK and HSC‐3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC‐3 BRAK cells than in HSC‐3 Mock cells. Also, HSC‐3 BRAK cells showed more rapid adhesion than HSC‐3 Mock cells when cultured on type I collagen‐coated dishes but not on fibronectin or laminin 1‐coated ones. This adhesion was mediated by α2β1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC‐3 BRAK cells formed much more elongated focal adhesions co‐localized with paxillin and actin stress fibres than did HSC‐3 Mock cells. Treatment of parental HSC‐3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC‐3 Mock and HSC‐3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC‐3 BRAK cells was perturbed by the introduction of an expression vector of the Rap‐binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC‐3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC‐3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.     

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non‐SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non‐SP cells. From a cell regulation point of view, we showed that SP activity depended on O₂ concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia‐induced factors HIF‐1α and HIF‐2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non‐SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.     

Encapsulated cells producing retroviral vectors for in vivo gene transfer

Background

Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated.

Methods

Vector (pBAG)‐producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X‐gal staining, as well as for the release of particles by performing RT‐PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for β‐galactosidase activity and by RT‐PCR.

Results

In culture, the virus‐producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self‐limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus‐producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC‐identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co‐implanted capsules.

Conclusion

The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene Transfer Approaches to the Lysosomal Storage Disorders

The work summarized in this paper used animal and cell culture models systems to develop gene therapy approaches for the lysosomal storage disorders. The results have provided the scientific basis for a clinical trial of gene transfer to hematopoietic stem cells (HSC) in Gaucher disease which is now in progress. The clinical experiment is providing evidence of HSC transduction, competitive engraftment of genetically corrected HSC, expression of the GC transgene, and the suggestion of a clinical response. In this paper we will review the progress made in Gaucher disease and include how gene transfer might be studied in other lysosomal storage disorders.     

Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice

Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation.