Youngtae Hong

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.

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 viurs (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.

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

As an alternative method to the conventional therapies for Hunters 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.

Transactivation activity of the human cytomegalovirus IE2 protein occurs at steps subsequent to TATA box-binding protein recruitment

The IE2 protein of human cytomegalovirus transactivates viral and cellular promoters through a wide variety of cis-elements, but the mechanism of its action has not been well characterized. Here, IE2-Sp1 synergy and IE2-TATA box-binding protein (TBP) interaction are examined by artificial recruitment of either Sp1 or TBP to the promoter. It was found that IE2 could cooperate with DNA-bound Sp1. A 117 amino acid glutamine-rich fragment of Sp1, which can interact with Drosophila TAF(II)110 and human TAF(II)130, was sufficient for the augmentation of IE2-driven transactivation. In binding assays in vitro, IE2 interacted directly with the C-terminal region of Sp1, which contains the zinc finger DNA-binding domain, but not with its transactivation domain, suggesting that synergy between IE2 and the transactivation domain of Sp1 might be mediated by other proteins such as TAF or TBP. It was also found that TBP recruitment to the promoter markedly increased IE2-mediated transactivation. Thus, IE2 acts synergistically with DNA-bound Sp1 and DNA-bound TBP. These results suggest that, in human cytomegalovirus IE2 transactivation, Sp1 functions at an early step such as recruitment of TBP and IE2 acts to accelerate rate-limiting steps after TBP recruitment.

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

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.

Sequences downstream of the RNA initiation site of the HTLV type I long terminal repeat are sufficient for trans-activation by human cytomegalovirus immediate-early proteins.

Human T cell leukemia virus type I infection is associated with a low incidence of morbidity in the form of adult T cell leukemia and neurologic disease, suggesting that there are other factors determining the pathogenic outcome of infection. We found that HCMV could infect various human cell lines known to be susceptible to HTLV-I infection, including T cell lines already harboring HTLV-I, and that HCMV infection could highly activate gene expression from the HTLV-I LTR. In addition, the coexpression of IE1 and IE2 genes of HCMV increased transcription from the HTLV-I LTR. The deletion analysis indicated that the entire U3 region is not required, but that the 216-bp region from +101 to +316 is sufficient for activation of the LTR by IE1 and IE2. These results suggest that HCMV IE proteins may affect the level of HTLV-I gene expression in coinfected individuals by interacting with HTLV-I LTR sequences.

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 cisacting 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. *** DIRECT SUPPORT *** A00RC002 00014

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

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.

104. Stable High Level Gene Expression by Minimum Sized Murine Leukemia Virus (MLV)-Based Retroviral Vectors in the Mouse Model

Top of pageAbstract Despite the frequent use of MLV-based retroviral vectors in gene therapy trials, there have been three major criticisms, often relative to the lentivirus-based retroviral vector. First is the claim that MLV-based vector drives a lower level and shorter period gene expression in vivo. However, there is no firm basis for this claim as there have been no serious comparative experiments done thus far between two retroviral vectors. Second, MLV-based vectors cannot deliver a gene to the resting cells. Although this currently holds true, considering the given speed of progress in cell biological technique, the number of cell type to which MLV-based vector can deliver the gene would increase. Third is the possibility of insertional mutagenesis. However, it should not be considered any disadvantage over lentiviral vector, because it also integrates into the host genome like any other retroviruses. Contrary to what has been perceived in the community, MLV-based vectors still have distinctive advantages over lentiviral vectors, especially when the easiness of overall handling, including the production process, is concerned. To study how MLV-based vectors perform in vivo, we tested a series of vectors derived from MT in mouse model. MT is a minimum sized vector that contains no viral coding sequences, but can efficiently drive gene expression and produce high viral titer (Kim et al, J. Virol, 1998, 994-1004; Yu et al, J. Virol. 2000, 8775-8780; Gene Ther., 2000, 797-804; Hong et al, J. Gen. Med., 2003, 18-29). Bone marrow (BM) cells were isolated from mice, and Lin- cells were enriched and transduced with MT-based vectors expressing GFP. 1 |[times]|105 Lin- cells were pre-stimulated with a specially formulated medium for 48 hours and transduced three times at MOI=5 over the period of another two days. At the end, 50|[ndash]|60% of these BM-derived cells became positive to GFP. Immediately after the gene delivery procedure, 1|[times]|106 cells were injected into the lethally irradiated recipient mice through the tail vein (i.v.) or the BM directly (i.BM). During the 40 weeks of follow-up analysis, some differences were found between i.v. (14|[sim]|23%) and i.BM (5|[sim]|14%) injections in terms of the percentage of GFP+ cells in PB cells. Our in vivo comparative analysis indicated that the vector containing the MSCV LTR gave a higher GFP expression than the one with the MoMuLV LTR. Similar long term gene expression was also observed in the mouse model using a MoMuLV-LTR driven vector expressing gp91 (whose defect leads to chronic granulomatous disease in humans). When the integration sites of MT-based vector were analyzed in transduced cells, it appears that there is no strong preference for any particular site as expected from the previous reports. Our data indicated that MLV-based vectors can drive high level gene expression during a substantially long period of time. Given the fact that an MLV-based vector is the most scrutinized gene delivery vehicle with a proven record of safety and efficiency, it should remain a strong candidate as a gene transfer system in the actual clinical settings.

396. Factors Affecting Retroviral Gene Transfer to Human CD34+ Cells

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. We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on 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 that used in medium formulation, could be replaced with human serum or synthetic material. 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 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. Our data could be useful in designing the procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.