Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial.

X-linked chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by a defect in the gp91(phox) gene. In an effort to treat X-CGD, we investigated the safety and efficacy of gene therapy using a retroviral vector, MT-gp91. Two X-CGD patients received autologous CD34(+) cells transduced with MT-gp91 after a conditioning regimen consisting of fludarabine and busulfan. The level of gene-marked cells was highest at day 21 (8.3 and 11.7% in peripheral blood cells) but decreased to 0.08 and 0.5%, respectively, 3 years after gene transfer. The level of functionally corrected cells, as determined by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, reached a peak at day 17 (6.5% patient 1 (P1) and 14.3% patient 2 (P2) of total granulocytes) and declined to 0.05% (P1) and 0.21% (P2), 3 years later. Some retroviral vectors were found to have integrated within or close to the proto-oncogenes MDS1-EVI1, PRDM16, and CCND2; however, no abnormal cell expansion or related hematological malignancy was observed. Overall, the gene transfer procedure did not produce any serious adverse effects and was able to convert a significant fraction of blood cells to biologically functional cells, albeit for a short period of time.

The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase.

Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments.

Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector.

BACKGROUND: The use of retroviral vectors has shown an actual clinical benefit in a few inherited diseases. However, the occurrence of cases of leukemia after the X-SCID gene therapy trial raised concerns about the safety of insertional mutagenesis inherent to the biology of the retrovirus. Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon. METHODS: In the present study, we report an in vitro assay system in which the effect of retroviral integration on the expression of the neighbouring gene can be studied. In this system, a retroviral vector and the neighbouring reporter gene were constructed in a single plasmid as if it had integrated into the chromosome. RESULTS: Using this assay, we found that the full-length long terminal repeat (LTR) could indeed activate the neighbouring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, whereas the truncated LTR exerted little influence. CONCLUSIONS: This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.

High efficiency gene transfer to human CD34+ cells.

We have previously reported the development of improved MLV-based retroviral vectors whose prototype is entitled MT (Kim et al, J. Virol. 72:994-1044; Yu et al, Gene Therapy 7:797-804). The MT vector does not contain any viral coding sequences, and thus the possibility of homologous recombination between the vector and the packaging genome is virtually nil. Indeed, in a shotgun RCR detection assay, an MT-based vector did not produce any RCR. On the contrary, the MFG vector, containing parts of all three viral coding sequences (gag, pol, and env), generated a significant number of RCR. In addition to being safe, MT-based vectors produce levels of gene expression and viral titer comparable to or higher than other vectors currently available within the community. Based on this vector, we have constructed a number of retroviral vectors that can be used for the treatment of a variety of human diseases. Our major target diseases are those that can be treated with or the status of which can be significantly improved with bone marrow transplantation. To obtain the most significant therapeutic effects, it is necessary to achieve the highest possible gene delivery efficiency, drive the highest level of gene expression, and prevent expression of the inserted therapeutic gene from being negatively influenced by the genome environment. To these ends, we compared various LTRs for their effects on the level of gene expression, tested the effect of cis-acting elements that may influence chromatin structure or position effect of the inserted gene, and studied different transduction conditions for their gene delivery efficiency. Data recently obtained from these experiments will be presented.

Construction of a retroviral vector production system with the minimum possibility of a homologous recombination.

A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia virus (MLV)-based packaging constructs contained significantly long 5' and/or 3' untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed.

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells.

BACKGROUND: Retrovirus-mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest. METHODS: We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material. RESULTS: Formulation A, consisting of stem cell factor, Flt-3 ligand, thrombopoietin, and IL-3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood. CONCLUSIONS: Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.

Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome.

BACKGROUND: As an alternative method to the conventional therapies for Hunter's syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector. METHODS: We constructed three types of MLV-based retroviral vectors expressing iduronate-2-sulfatase (IDS) which is deficient in patients suffering from Hunter's syndrome: MIN-IDS and MIM-IDS, which express IDS along with bacterial neo and human MDR genes, respectively, and MT-IDS lacking any selectable marker. Respective producer lines were derived from the packaging line, PG13, and compared for viral titer and levels of gene expression. After comparing, the retroviral vector, MT-IDS, was used to transduce human bone marrow CD34+ stem cells on fibronectin under various MOIs. RESULTS: In comparison, MT-IDS not only produced the highest viral titer (close to 10(7) cfu/ml), but also showed the highest level of gene expression in various transduction assays and RNA analysis. When 1.5 x 10(5) human CD34+ bone marrow cells were transduced with MT-IDS under the most optimal MOIs, about 80% of total colony forming units were shown to contain the IDS cDNA. CONCLUSIONS: Minimum-sized retroviral vector that contains no selective marker as well as a viral coding sequences could drive a high level of gene expression, be produced efficiently from the producer line, and enter hematopoietic cells at a high frequency. Our data suggest the great potential for using MT-based vector(s) in a gene therapy trial for Hunter's syndrome utilizing human CD34+ stem cells as target cells.