Karim Lee

Acquisition of HIV-1 resistance in T lymphocytes using an ACA-specific E. coli mRNA interferase.

Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.

Downregulation of GFAP, TSP-1, and p53 in Human Glioblastoma Cell Line, U373MG, by IE1 Protein From Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a member of the β‐herpesvirus family, which has tropism for glial cells. It was recently reported that HCMV might play important roles in the pathogenesis of malignant glioma. In this study, we investigated the effects of the HCMV IE1 protein on the gene expression profile in the human glioblastoma cell line, U373MG by employing cDNA microarray technology. Using DNA chips containing approximately 1,000 human cDNAs, RNA samples from U373MG cells stably expressing IE1 were compared with those from the control cells lacking IE1 cDNA. Fluorescence intensities of 13 genes were significantly decreased in IE1‐expressing cells, while one gene was found to be upregulated. Among these 14 genes, we chose to work further on glial fibrillary acidic protein (GFAP), thrombospondin‐1 (TSP‐1), and p53, because of their previously known involvement in tumorigenesis. The mRNA levels of all these genes were found to be decreased in IE1‐expressing glioblastoma cells by real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) as well as Northern blot analysis. The decreased expression of these genes was also observed at protein levels as measured by immunocytochemistry or fluorescence‐activated cell sorting (FACS) analysis. Our data strongly suggested that HCMV IE1 could modulate the expression of cellular genes that might play important roles in the pathogenesis of glial tumors.

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.

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.

Human cytomegalovirus infection downregulates the expression of glial fibrillary acidic protein in human glioblastoma U373MG cells: identification of viral genes and protein domains involved.

Human cytomegalovirus (HCMV) has tropism for glial cells, among many other cell types. It was reported previously that the stable expression of HCMV immediate-early protein 1 (IE1) could dramatically reduce the RNA level of glial fibrillary acidic protein (GFAP), an astroglial cell-specific intermediate filament protein, which is progressively lost with an increase in glioma malignancy. To understand this phenomenon in the context of virus infection, a human glioblastoma cell line, U373MG, was infected with HCMV (strain AD169 or Towne). The RNA level of GFAP was reduced by more than 10-fold at an m.o.i. of 3 at 48 h post-infection, whilst virus treated with neutralizing antibody C23 or with UV light had a much-reduced effect. Treatment of infected cells with ganciclovir did not prevent HCMV-mediated downregulation of GFAP. Although the expression of GFAP RNA is downregulated in IE1-expressing cells, a mutant HCMV strain lacking IE1 still suppressed GFAP, indicating that other IE proteins may be involved. IE2 is also proposed to be involved in GFAP downregulation, as an adenoviral vector expressing IE2 could also reduce the RNA level of GFAP. Data from the mutational analysis indicated that HCMV infection might affect the expression of this structural protein significantly, primarily through the C-terminal acidic region of the IE1 protein.

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 murine leukemia virus-based retroviral vectors with a minimum possibility of cis-activation

The possibility of insertional mutagenesis in retroviral gene therapy can be reduced by using a vector lacking the enhancer sequence present in the U3 of the long-terminal repeats. However, such vectors suffer from many pitfalls. We attempted to improve a murine leukemia virus-based retroviral vector containing the enhancer-free U3, first by making it easier to construct a producer line and then by introducing the cellular RPL10 promoter as an internal promoter. The reverse orientation of the expression cassette of the transgene was found to give higher transducing titer and higher-level gene expression. The deletion analysis revealed that the 54-bp-long sequence of U3 (34 and 20 bp present at 5′ and 3′ extreme ends, respectively) was sufficient for the functions of retroviral vectors. The data from the in vitro cell culture assay indicated that the final construct, ROK, containing all these features, had little cis-activation activity, even if it was placed right upstream from the RNA start site of the neighboring gene. Our data suggested that the newly developed vector might provide increased safety, while still producing high viral titer from a stable producer line and high-level gene expression in various target cells including human CD34+ stem cells.

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.