Epidermal stem cells as targets for gene transfer

One of the characteristics of the epidermis that makes it an attractive tissue for gene therapy is that it is renewed through proliferation of stem cells. If the stem cells can be transduced with the gene of interest then expression of that gene should continue throughout adult life. This article discusses current research on epidermal stem cells, highlighting progress in their identification and in discovering the mechanisms that regulate exit from the stem cell compartment.     

The potential of cord blood stem cells for use in regenerative medicine

It is estimated that up to 128 million individuals might benefit from regenerative medicine therapy, or almost 1 in 3 individuals in the US. If accurate, the need to relieve suffering and reduce healthcare costs is an enormous motivator to rapidly bring stem cell therapies to the clinic. Unfortunately, embryonic stem (ES) cell therapies are limited at present by ethical and political constraints and, most importantly, by significant biologic hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend on the development of non-ES cell therapies. At present, non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). Each of these stem cells is being used to treat a variety of diseases. This review shows that CB contains multiple populations of pluripotent stem cells, and can be considered the best alternative to ES cells. CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopedic, neurologic and endocrine diseases.     

The potential of cord blood stem cells for use in regenerative medicine

It is estimated that up to 128 million individuals might benefit from regenerative medicine therapy, or almost 1 in 3 individuals in the US. If accurate, the need to relieve suffering and reduce healthcare costs is an enormous motivator to rapidly bring stem cell therapies to the clinic. Unfortunately, embryonic stem (ES) cell therapies are limited at present by ethical and political constraints and, most importantly, by significant biologic hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend on the development of non-ES cell therapies. At present, non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). Each of these stem cells is being used to treat a variety of diseases. This review shows that CB contains multiple populations of pluripotent stem cells, and can be considered the best alternative to ES cells. CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopedic, neurologic and endocrine diseases.     

Gene therapy progress and prospects: gene therapy for severe combined immunodeficiency

Severe combined immunodeficiencies have long been targeted as a group of disorders amenable to gene therapy because of their defined molecular biology and pathophysiology, and the prediction that corrected cells would have profound growth and survival advantage. Recently, several clinical studies have shown that conventional gene transfer technology can produce major beneficial therapeutic effects in these patients, but, as for all cellular and pharmacological treatment approaches, with a finite potential for toxicity.     

Progress in the use of gene transfer methods to treat genetic blood diseases

A report by French physician-scientists suggests a successful application of gene transfer methods in the treatment of two children with severe combined immunodeficiency (SCID) due to defective interleukin 2 receptor common gamma chain. The protocol used in this clinical trial was derived from a number of preclinical and basic studies leading to improved transduction of hematopoietic stem and primitive progenitor cells using retrovirus vectors. These improvements have also been shown to impact transduction of a long-lived progenitor cell in a chemotherapy protocol in cancer patients. The improved results of these human trials come during a period of increased scrutiny and criticism of human gene therapy trials, due, in part, to significant toxicities in some trials using adenovirus-based vectors. The potential efficacy versus toxicity of phase I trials of human gene therapy is also under question. After many years of research, however, there appears to be real evidence that genetic diseases may be successfully treated by gene transfer techniques. Future clinical studies should be based on continued progress in the understanding of the toxicology of gene delivery systems, vector technology, and target cell manipulation.     

Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency

Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010 ). Currently, rAAV vectors are being utilized in phase I/II clinical trials for cystic fibrosis, α-1 antitrypsin deficiency, Canavan's disease, Parkinson's disease, hemophilia, limb-girdle muscular dystrophy, arthritis, Batten's disease, and Leber's congenital amaurosis (Flotte et al., 1996 , 2004 ; Kay et al., 2000 ; Aitken et al., 2001 ; Wagner et al., 2002 ; Manno et al., 2003 ; Snyder and Francis, 2005 ; Maguire et al., 2008 ; Cideciyan et al., 2009 ). In this study, we present preclinical data to support the viability of an rAAV-based gene transfer strategy for cure of ADA-SCID. We report efficient transduction of a variety of postmitotic target tissues in vivo, subsequent human ADA (hADA) expression, and enhanced hADA secretion in tissues and blood, with increasing peripheral lymphocyte populations over time.     

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.     

Heterogeneity of b lymphocyte differentiation in severe combined immunodeficiency disease.

Pokeweed mitogen-induced B lymphocyte differentiation in vitro into antibody secreting plaque-forming cells (PFC) was investigated in nine patients with severe combined immunodeficiency having variable proportions of circulating B lymphocytes. When cultured by themselves, the peripheral blood mononuclear cells did not respond to stimulation with pokeweed mitogen in any patient. In the presence of irradiated allogeneic T cells as helpers, however, PFC responses were elicited in lymphocyte cultures from peripheral blood and/or bone marrow in some patients. In one of these patients, results of allogeneic co-culture experiments were suggestive of genetically restricted suppressor cells. In a single patient with deficiency of the enzyme adenosine deaminase, PFC were generated in bone marrow lymphocyte cultures only when they were supplemented with exogenous adenosine deaminase and allogeneic helper cells. A parallel study of T lymphocyte differentiation in vitro performed in fractionated bone marrow cells was suggestive of arrested differentiation at different steps along the differentiation pathway. In two patients with evidence of functional B cell precursors, deficiencies of helper T cell function could be attributed to differentiation defects at the level of the stem cells in one and the thymus in the other. The findings reported here further substantiate the heterogeneity of the severe combined immunodeficiency disease syndromes.     

Molecular genetic analysis of herpesviruses and their potential use as vectors for gene therapy applications

Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses. Some of these cells include neuronal cells and B-lymphocytes infected with herpes simplex virus (HSV) and Epstein-Barr virus (EBV), respectively as well as human T-lymphocytes with herpesvirus saimiri. These large DNA viruses have the potential to deliver fragments of human heterologous DNA > 150 kbp to specific cells. Additionally, EBV recombinants can maintain these large pieces of DNA in the infected B-cells as episomal DNA. The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 150 kbp and to infect cells that can stably maintain DNA as episomes. Moreover, other herpesvirus vector systems can be utilized as a source for the propagation of specific DNA fragments of interest, including wild-type genes to correct genetic defects in human cells. Other herpesvirus systems include defective infectious single cycle (DISC-HSV) mutants of HSV, which are now undergoing trials for the treatment of genital herpesvirus infections and certain types of cancers. The use of herpesviruses as an agent to deliver heterologous gene products has the potential to make a significant difference to the development of therapeutic approaches to human diseases.     

Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes

We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN+) was compared with spinoculation in noncoated tubes (spin/FN-) and transduction in plates coated with FN (plate/FN+). Antibody to TGF-beta was added to spin/FN+ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34+ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6+/-4.3, 9.1+/-1.6, and 21.1+/-6.5% of CD34+ cells in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. Transduction of CD+CD38low cells was also higher in the spin/FN+ arm as compared with transduction in the spin/FN- arm. These results were corroborated by colony-forming assays. Antibody to TGF-beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN+ as compared with the plate/FN+ and spin/FN-arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.     

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.     

T-lymphocyte differentiation in vitro in severe combined immunodeficiency. Defects of stem cells.

A study of T-lymphocyte differentiation was made on fractionated bone marrow cells from normal volunteers and from 11 patients with severe combined immunodeficiency (SCID) using normal thymic epithelial monolayers and their culture supernates as inducing agents. Normal marrow cells could regularly be induced to bear the human T-lymphocyte antigen (HTLA), to form rosettes with sheep erythrocytes (E rosettes), and to respond to the mitogen concanavalin A (Con A) after coculture with the thymic epithelial monolayers or their culture supernates. In contrast, studies of T-cell differentiation on the marrow cells of patients with SCID revealed varying defects, ranging from a complete "absence" of definable T-cell precursors to partial differentiation resulting in acquisition of one (HTLA) or two (HTLA and E rosettes) markers for T lymphocytes. Only in one patient was there induction of all three T-cell markers, namely, HTLA, E rosettes, and responsiveness to Con A. These observations indicate that SCID is a heterogeneous disorder in which defects of differentiation can occur at one or more multiple sites of differentiation leading the the clinical expression of T- and B-cell dysfunction. Further, our studies indicate that in T-cell differentiation, HTLA probably appears before the capacity to form E-rosettes, and development of the latter capacity is followed by a state of responsiveness to mitogens. A scheme of normal differentiation along with the defects of precursor T cells seen in SCID is presented.     

Toward the use of endometrial and menstrual blood mesenchymal stem cells for cell-based therapies

Introduction: Bone marrow is a widely used source of mesenchymal stem cells (MSCs) for cell-based therapies. Recently, endometrium – the highly regenerative lining of the uterus – and menstrual blood have been identified as more accessible sources of MSCs. These uterine MSCs include two related cell types: endometrial MSCs (eMSCs) and endometrial regenerative cells (ERCs).

Areas covered: The properties of eMSCs and ERCs and their application in preclinical in vitro and in vivo studies for pelvic organ prolapse, heart disorders and ischemic conditions are reviewed. Details of the first clinical Phase I and Phase II studies will be provided.

Expert opinion: The authors report that eMSCs and ERCs are a readily available source of adult stem cells. Both eMSCs and ERCs fulfill the key MSC criteria and have been successfully used in preclinical models to treat various diseases. Data on clinical trials are sparse. More research is needed to determine the mechanism of action of eMSCs and ERCs in these regenerative medicine models and to determine the long-term benefits and any adverse effects after their administration.     

人混合脐血对SCID小鼠移植实验研究

目的：探讨人混合脐血移植的特性。方法：将人单个脐血或两个ＨＬＡ不相合的混合脐血输入经亚致死剂量照射后的严重联合免疫缺陷（ＳＣＩＤ）小鼠，观察两组脐血在ＳＣＩＤ小鼠体内的植入率和ＳＣＩＤ小鼠的造血重建及免疫功能的特性。结果：在输入造血干／祖细胞总量相等时，两组移植成功率无明显差异（Ｐ＞０．０５）；混合脐血组的植入率较单个脐血组的低［人ＣＦＵ－ＧＭ为２．８０±１．７９／２×１０５ＭＮＣｓ比１３．４０±８．１４／２×１０５ＭＮＣｓ（Ｐ＜０．０５）；ＣＤ４５＋细胞为０．９１％±０．６０％比８．９６％±１．７９％（Ｐ＜０．０１）］；混合脐血组ＳＣＩＤ小鼠的淋巴细胞转化率也较低（４．００％±４．１８％比２４．６０％±１８．３２％，Ｐ＜０．０５）。用多聚酶链反应－寡聚核苷酸单链构象多态性检测人ＨＬＡ－ＤＰＢ基因发现，两个脐血混合移植只有１个脐血植入ＳＣＩＤ小鼠，该脐血含造血干／祖细胞数（ＣＦＵ－ＧＭ、ＣＤ３４＋细胞）较多。结论：ＨＬＡ不相合的两个脐血混合移植ＳＣＩＤ小鼠可以重建其造血和免疫功能，但仅有含造血干／祖细胞较多、增殖活性较高的那个脐血植入ＳＣＩＤ小鼠。     

Epstein-Barr virus vector-mediated gene transfer into human B cells: potential for antitumor vaccination

The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vector-derived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas.