Jong-Mook Kim

Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease

This phase 1 clinical trial tested the safety of intramuscular gene transfer by using naked plasmid DNA encoding the gene for VEGF, and analyzed the potential therapeutic benefits in patients with severe peripheral arterial disease (PAD). This study was an open-labeled, dose- escalating, single-center trial on nine male patients with severe debilitating PAD who had not responded to conventional therapy. Seven had Buergers disease and two had arteriosclerosis obliterans. Plasmid DNA (pCK) containing human VEGF165 was given by eight intramuscular injections in and around the area in need of new blood vessels. The study evaluated three escalating total doses (2, 4, and 8 mg of pCK- VEGF165), with half of each total dose given four weeks apart. The follow-up duration was nine months. The gene injections were well tolerated without significant side effects or laboratory abnormalities related to gene transfer. Three patients showed transient edema in their extremities. Ischemic pain of the affected limb was relieved or improved markedly in six of seven patients. Ischemic ulcers healed or improved in four of six patients. The mean ankle-brachial index (ABI) improved significantly. Six of nine patients showed an increase in collateral vessels around the injection sites demonstrated by digital subtraction angiography. However, there was no relationship between the degree of ABI improvement and the dose given. Mean plasma levels of VEGF did not increase significantly. In conclusion, intramuscular injections of pCK- VEGF165 can be performed safely to induce therapeutic angiogenesis in patients with severe PAD.

A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia.

The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK‐HGF‐X7) in critical limb ischemia (CLI) patients.

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL‐1Ra for the prevention of murine collagen‐induced arthritis (CIA).

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.

The 17 Nucleotides Downstream from the env Gene Stop Codon Are Important for Murine Leukemia Virus Packaging

ABSTRACT We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of theenv gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5′ long terminal repeat (LTR) and the other immediately upstream from the 3′ LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed.

α-Galactosylceramide-loaded, antigen-expressing B cells prime a wide spectrum of antitumor immunity

Most of the current tumor vaccines successfully elicit strong protection against tumor but offer little therapeutic effect against existing tumors, highlighting the need for a more effective vaccine strategy. Vaccination with tumor antigen‐presenting cells can induce antitumor immune responses. We have previously shown that NKT‐licensed B cells prime cytotoxic T lymphocytes (CTLs) with epitope peptide and generate prophylactic/therapeutic antitumor effects. To extend our B cell vaccine approach to the whole antigen, and to overcome the MHC restriction, we used a nonreplicating adenovirus to transduce B cells with antigenic gene. Primary B cells transduced with an adenovirus‐encoding truncated Her‐2/neu (AdHM) efficiently expressed Her‐2/neu. Compared with the moderate antitumor activity induced by vaccination with adenovirus‐transduced B cells (B/AdHM), vaccination with α‐galactosylceramide‐loaded B/AdHM (B/AdHM/αGalCer) induced significantly stronger antitumor immunity, especially in the tumor‐bearing mice. The depletion study showed that CD4+, CD8+ and NK cells were all necessary for the therapeutic immunity. Confirming the results of the depletion study, B/AdHM/αGalCer vaccination induced cytotoxic NK cell responses but B/AdHM did not. Vaccination with B/AdHM/αGalCer generated Her‐2/neu‐specific antibodies more efficiently than B/AdHM immunization. More importantly, B/AdHM/αGalCer could prime Her‐2/neu‐specific cytotoxic T cells more efficiently and durably than B/AdHM. CD4+ cells appeared to be necessary for the induction of antibody and CTL responses. Our results demonstrate that, with the help of NKT cells, antigen‐transduced B cells efficiently induce innate immunity as well as a wide range of adaptive immunity against the tumor, suggesting that they could be used to develop a novel cellular vaccine.

Enhanced cardioprotective effects by coexpression of two isoforms of hepatocyte growth factor from naked plasmid DNA in a rat ischemic heart disease model

The therapeutic potential of pCK‐HGF‐X7, a naked DNA designed to express two isoforms of hepatocyte growth factor (HGF723 and HGF728), was studied in the rat ischemic heart disease model.

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.

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis.

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (Cmax) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.

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.