Adeno-associated virus-mediated gene transfer in hematopoietic stem/ progenitor cells as a therapeutic tool

Hematopoietic stem cells (HSCs) have unique properties of self-renewal, differentiation and proliferation. HSCs are easily accessible, and can be readily delivered back to patients by autologous transplantation, which renders them as attractive targets for ex vivo gene therapy. The adeno-associated virus (AAV) vectors have to date not been associated with any malignant disease, and have gained attention as a potentially safer alternative to the more commonly used retroviral vectors for HSC gene therapy. Although conflicting data exist with regard to HSC transduction by AAV vectors, in this review, we provide an overview of AAV-mediated HSC gene transfer - obstacles as well strategies to improve the transduction efficiency - and the potential use of AAV vectors for gene therapy of human diseases involving HSCs.     

腺相关病毒载体介导造血干/祖细胞的基因转导

造血干细胞 (HSCs)具有自我更新、分化和增殖的独特特性，并可用自体移植法移植给病人,这使其成为基因治疗中引人注目的靶点。到目前为止尚未发现腺相关病毒（AAV）载体与任何恶性疾病有明确的关系。在利用造血干细胞的基因治疗中，AAV载体受到人们的广泛关注。既往的AAV转导HSCs的研究结果不完全一致,现将该方面的研究进展作一概述。     

Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease

Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.     

Advances in the treatment of Chronic Granulomatous Disease by gene therapy

Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we review the developments leading to a gene therapy approach for the treatment of Chronic Granulomatous Disease (CGD), a primary life threatening immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Although the disease can be cured by bone marrow transplantation, this treatment is only available to patients with HLA-identical sibling or matched unrelated donors. One therapeutic option for patients without suitable donor is the genetic modification of autologous hematopoietic stem cells. Although early attempts to correct CGD by gene therapy were unsuccessful, these studies demonstrated the safety and limitations of gene transfer into hematopoietic stem cells (HSC) of CGD patients using retroviral vectors. The recent development of advanced gene transduction protocols together with improved retroviral vectors, combined with low intensity chemotherapy conditioning, allowed partial correction of the granulocytic function with a significant clinical benefit in treated patients. These results may have important implications for future applications of gene therapy in myeloid disorders and inherited diseases using hematopoietic stem cells.     

Safety concerns related to hematopoietic stem cell gene transfer using retroviral vectors

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.     

Human hematopoietic stem cells in gene therapy: pre-clinical and clinical issues

Hematopoietic stem and progenitor cells (HSC) have been widely used in allogeneic transplant procedures, therefore their intrinsic characteristics, the biology of their niche in the bone marrow, and the mobilization and homing processes have been extensively investigated. With the development of gene therapy strategies, new therapeutic options based on autologous HSC have become available which may reduce the morbidity and mortality associated to allogeneic transplantation, but require an ex vivo manipulation of the cells to be corrected before re-infusion. For the success of these approaches it is necessary to optimize culture conditions in order to achieve efficient cell transduction while preserving the biological properties of the stem cells. We review here the factors critical for achieving efficient HSC transduction and maintenance of HSC stemness and homing capacity upon ex vivo culture. When HSC gene therapy is used in genetic disorders, permanent integration of therapeutic genes into the chromosomes of affected cells is needed. Indeed, by use of integrating vectors, such as retroviruses, gene therapy has met significant success in immunodeficiency syndromes characterized by a selective advantage of the transduced cells. However, retroviral integration can take place in stem cells at a variety of chromosomal sites, and examples have been reported of integration of therapeutic vectors causing cancer in patients. The clinical benefit arising from the long-term correction of the genetic defect, due to vector integration into the HSC genome, and the adverse consequences of these events are also here discussed, together with the new and challenging perspectives of HSC gene therapy.     

Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration

Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.     

High efficiency and long-term foreign gene expression in cultured liver sinusoidal endothelial cells by retroviral transduction.

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.     

T lymphocytes as targets of gene transfer with Moloney-type retroviral vectors

Peripheral T lymphocytes are a target of choice for many gene therapeutic strategies. Retrovirus-mediated transduction allows genomic integration and long-term expression of transgenes in target cells. Over many years, low transduction efficiency into primary T lymphocytes has limited clinical application of existing protocols. Recently, gene transfer rates > 50% have been achieved facilitating clinical studies. More attention is thus being focused on the ability of gene-modified cells to carry out innate as well as conferred functions in vivo and the influence of culture conditions, retroviral vector and host response thereon.     

Retrovirus-mediated gene transfer into human hematopoietic stem cells

 Human hematopoietic stem cells genetically modified by retroviral-mediated gene transfer may offer new treatment options for patients with genetic disease. The potential of gene-modified hematopoietic stem cells as vehicles for gene delivery was first illustrated by the demonstration that hematopoietic systems of lethally irradiated mice can be reconstituted with retroviral vector transduced syngeneic bone marrow, and that these cells can in turn provide genetically marked progeny which persist in blood and marrow over extended time periods [1–4]. In contrast, hematopoietic stem cells from large animals prove difficult to transduce with retroviral vectors and are consequently less likely to function as vehicles for long-term gene therapy. Indeed, clinically relevant levels of gene transfer into large animal and human hematopoietic stem cells has not been widely achieved. The need for improved retroviral vector systems and for understanding the biology of hematopoietic stem cell gene transfer continue to fuel intense research activity. Preliminary results from human stem cell gene marking and gene therapy trials currently underway are encouraging. This contribution reviews the underlying concepts relevant to retroviral-mediated gene transfer into hematopoietic stem cells. We survey the evolution of approaches for gene transfer into hematopoietic stem cells, from murine and large animal models to the first human clinical trials. Finally, we discuss new strategies which are currently being pursued. Received: 12 March 1997 / Accepted: 21 July 1997     

Use of VSV-G pseudotyped retroviral vectors to target murine osteoprogenitor cells

Marrow stromal cells (MSC) and neonatal calvarial cells have the potential to differentiate and express markers of mature osteoblasts. Furthermore, MSCs can generate multiple differentiated connective tissue phenotypes. These properties and their ability to be expanded ex vivo make them good models for ex vivo gene therapy. In this study we examined the ability of vesicular stomatitis virus (VSV-G) pseudotyped retroviral vectors to transduce osteoprogenitor cells derived from bone marrow and from neonatal calvaria. Retrovectors encoding either beta-galactosidase or green fluorescent protein (eGFP) were used for transduction of primary murine marrow stromal and primary neonatal calvarial cell cultures. High infection efficiency was demonstrated by fluorescence-activated cell analysis when GFP was used as a marker or by estimating the number of beta-galactosidase-positive cells. Expression of markers of differentiated bone cells, including Col1a1, bone sialoprotein, and osteocalcin mRNA and alkaline phosphatase activity was not impaired by retroviral transduction. Our data suggest that VSV-G pseudotypes retroviral vectors are suitable for introducing genes into osteoprogenitor cells without affecting osteoprogenitor lineage progression.     

New recombinant serotypes of AAV vectors

AAV based vectors can achieve stable gene transfer with minimal vector related toxicities. AAV serotype 2 (AAV2) is the first AAV that was vectored for gene transfer applications. However, the restricted tissue tropism of AAV and its low transduction efficiency have limited its further development as vector. Recent studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5 and 6 have shown improved potency and broadened tropism of the AAV vector by packaging the same vector genome with different AAV capsids. In an attempt to search for potent AAV vectors with enhanced performance profiles, molecular techniques were employed for the detection and isolation of endogenous AAVs from a variety of human and non-human primate (NHP) tissues. A family of novel primate AAVs consisting of 110 non-redundant species of proviral sequences was discovered and turned to be prevalent in 18-19% of the tissues evaluated. Phylogenetic and functional analyses revealed that primate AAVs are segregated into clades based on phylogenetic relatedness. The members within a clade share functional and serological properties. Initial evaluation in mouse models of vectors based on these novel AAVs for tissue tropism and gene transfer potency led to the identification of some vector with improved gene transfer to different target tissues. Gene therapy treatment of several mouse and canine models with novel AAV vectors achieved long term phenotypic corrections. Vectors based on new primate AAVs could become the next generation of efficient gene transfer vehicles for various gene therapy applications.     

Comparison of retroviral transduction efficiency in CD34+ cells derived from bone marrow versus G-CSF-mobilized or G-CSF plus stem cell factor-mobilized peripheral blood in nonhuman primates

Hematopoietic stem cells (HSCs) are ideal targets for genetic manipulation in the treatment of several congenital and acquired disorders affecting the hematopoietic compartment. Although G-CSF-mobilized peripheral blood CD34(+) cells are the favored source of hematopoietic stem cells in clinical transplantation, this source of stem cells does not provide meaningful engraftment levels of genetically modified cells compared with G-CSF + stem cell factor (SCF)-mobilized cells in nonhuman primates. Furthermore, the use of G-CSF mobilization can have disastrous consequences in patients with sickle cell disease, a long-held target disorder for HSC-based gene therapy approaches. We therefore conducted a study to compare the levels of genetically modified cells attainable after retroviral transduction of CD34(+) cells collected from a bone marrow (BM) harvest with CD34(+) cells collected from a leukapheresis product after mobilization with G-CSF (n = 3) or G-CSF in combination with SCF (n = 3) in the rhesus macaque autologous transplantation model. Transductions were performed using retroviral vector supernatant on fibronectin-coated plates for 96 hours in the presence of stimulatory cytokines. BM was equal to or better than G-CSF-mobilized peripheral blood as a source of HSCs for retroviral transduction. Although the highest marking observed was derived from G-SCF + SCF-mobilized peripheral blood in two animals, marking in the third originated only from the BM fraction. These results demonstrate that steady-state BM is at least equivalent to G-CSF-mobilized peripheral blood as a source of HSCs for retroviral gene transfer and the only currently available source for patients with sickle cell disease.     

Gene transfer into hematopoietic stem cells using lentiviral vectors

Gene transfer into hematopoietic cells is currently being used to modulate immune responses, to protect hematopoietic cells against cytotoxic drugs or viral genes, and to restore gene deficiencies due to either inborn genetic defects or acquired loss of regular gene function. In particular, gene addition strategies for inherited severe combined immunodeficiencies (SCID) due to adenosine deaminase (ADA) deficiency or defects of the interleukin-2 receptor gamma-chain represent potentially curative strategies based on gene transfer into hematopoietic cells using recombinant retroviral vectors. Since long-term correction of genetic defects in hematopoietic cells often requires transduction of hematopoietic stem cells, an effective gene transfer into stem cells with efficient long-term and multi-lineage transgene expression is the desired goal for these therapeutic strategies. However, gene transfer strategies with retroviral vectors unable to integrate into non-cycling cells are limited by the quiescent state of the stem cells that have to be stimulated by cytokines to induce cell cycle progression. To circumvent these barriers, lentiviral vector systems based on HIV-1 have recently been developed which are able to deliver and express genes in non-dividing cells both in vitro and in vivo. This review outlines the development and improvement of lentivirus-based gene transfer protocols and discusses the use of lentiviral vectors in preclinical gene therapy studies.     

Construction of retroviral vectors with enhanced efficiency of transgene expression

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.     

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction

BACKGROUND AND METHODS. Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS. Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS. These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.     

Differential interaction of retroviral vector with target cell: quantitative effect of cellular receptor, soluble proteoglycan, and cell type on gene delivery efficiency

Retroviral vectors are powerful tools for gene therapy and stem cell engineering. To improve efficiency of retroviral gene delivery, quantitative understanding of interactions of a retroviral vector and a cell is crucial. Effects of nonspecific adsorption of retrovirus on a cell via proteoglycans and receptor-mediated binding of retrovirus to a cell on overall transduction efficiency were quantified by combining a mathematical model and experimental data. Results represented by transduction rate constant, a lumped parameter of overall transduction efficiency, delineated that chondroitin sulfate C (CSC) plays dual roles as either enhancer or inhibitor of retroviral transduction, depending on its concentrations in the retroviral supernatant. At the concentration of 20 microg/mL, CSC enhanced the transduction efficiency up to threefold but inhibited more than sevenfold at the concentration of 100 microg/mL. Transduction rate constants for amphotropic retroviral infection of NIH 3T3 cells under phosphate-depleted culture condition showed a proportional relationship between cellular receptor density on a cell and transduction efficiency. It was finally shown that amphotropic retrovirus transduced human fibroblast HT1080 cells more efficiently than NIH 3T3 cells. On the contrary, the transduction efficiency of NIH 3T3 cells by vesicular stomatitis virus G protein pseudotyped retroviruses was eightfold higher than that of HT1080 cells. This study implies usefulness of using quantitative analysis of retroviral transduction in understanding and optimizing retroviral gene delivery systems for therapeutic approaches to tissue engineering.     

Emerging adenoviral vectors for stable correction of genetic disorders

Recent drawbacks in treating patients with severe combined immunodeficiency disorders with retroviral vectors underline the importance of generating novel tools for stable transduction of mammalian cells. Substantial progress has been made over the recent years which may offer important steps towards stable and more importantly safer correction of genetic diseases. This article discusses recent advances for stable transduction of target cells based on adenoviral gene transfer. There is accumulating evidence that recombinant adenoviral vectors (AdVs) based on various human serotypes with a broad cellular tropism and adenoviruses (Ads) from different species will play an important role in future gene therapy applications. In combination with recombinant AdVs for somatic integration these gene transfer vectors offer high transduction efficiencies with potentially safer integration patterns. Other approaches for persistent transgene expression include excision of stable episomes from the adenoviral vector genome, but also long-term persistence of the complete adenoviral vector genome as an episomal DNA molecule was demonstrated and exemplified by the treatment of various genetic diseases in small and large animal models. This review displays advantages but also limitations of these Ad based vector systems. This is the perfect time to pursue such approaches because alternative strategies for stable transduction of mammalian cells undergoing many cell divisions are urgently needed. Looking into the future, we believe that a combination of different components from different viral vectors in concert with non-viral vector systems will be successful in designing significantly optimized transfer vehicles for a broad range of different genetic diseases.     

Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.