Xianmin Meng

Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy.

In vivo gene introduction into keratinocytes using jet injection

Successful keratinocyte gene therapy requires the development of efficient methods of gene transfer to keratinocytes. Jet injection of a solution containing DNA can be used to transfer genes to several tissues in vivo. In this article, we tried to introduce DNA into rat and human keratinocytes using this method. First, we fired a β-gal expression vector into rat skin at several distances using a jet injector and examined β-gal activity in the epidermal keratinocytes. The highest activity in keratinocytes was found when the plasmid was fired at 10 cm from the skin surface; the activity lessened as the firing distance became shorter than 10 cm. Next, we transplanted human skin on to a nude rat, fired the vector into the human skin from a distance of 10 cm and examined the β-gal activity. We also injected the same amount of plasmid with a needle to compare jet with needle injections. The results showed that jet injection of the naked DNA could introduce and express DNA in human keratinocytes in vivo and that jet injection exhibited much higher activity than needle injection. Jet injection of the naked DNA will provide a method for keratinocyte gene therapy in the future.

Relationship between the CYP2D6 genotype and the steady-state plasma concentrations of trazodone and its active metabolite m-chlorophenylpiperazine

Abstract The relationship between the cytochrome P450 (CYP) 2D6 genotype and the steady-state plasma concentrations (Css) of trazodone and its active metabolite m-chlorophenylpiperazine (mCPP) was studied in 54 depressed Japanese patients receiving trazodone 150 mg at bedtime. By use of allele-specific PCR analysis, the wild type allele, three mutated alleles causing absent enzyme activity (CYP2D6A, CYP2D6B and CYP2D6D) and one mutated allele causing decreased enzyme activity (CYPZD6 Ch) were identified. The means (ranges) of the Css of trazodone, corrected to the median body weight in 17 cases with no mutated allele, 27 cases with one mutated allele and 10 cases with two mutated alleles, were 556 (281–1115), 643 (302–1362) and 671 (234–1418) ng/ml, respectively, while the values of mCPP were 60 (35–121), 65 (33–99) and 58 (38–112) ng/ml, respectively. Neither the Css of trazodone (F = 0.80, P = 0.45) nor that of mCPP (F = 0.49, P = 0.61) significantly differed among the three groups. The present study thus suggests that the CYP2D6 genotype cannot predict the Css of these compounds.

Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin

Abstract: Using the plasmid DNA injection method, we introduced cytokine genes into skin to determine whether systemic expression of cytokine genes is possible. Eight human cytokine [interleukin‐4 (IL‐4), IL‐6, IL‐10, transforming growth factor β1 (TGF‐β1), monocyte chemotactic and activating factor (MCAF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumor necrosis factor α (TNF‐α) and interferon γ (IFN‐γ)] gene expression vectors were constructed and injected into rat skin. Transgenic cytokines in local keratinocytes and in the sera were assayed with ELISA. Our results showed that transgenic cytokines were markedly increased in keratinocytes at the injection site. The serum concentrations of IL‐4, 6, 10 and ΤGF‐β1 reached levels high enough to have systemic biologic effects. However, other cytokines used in this study could not be detected in the sera. Moreover, the serum transgenic IL‐10 level after subcutaneous injection was significantly higher than after intramuscular injection. We suggest that keratinocytes can be used as a bioreactor to achieve systemic expression of cytokine genes by DNA injection, but the transgenic protein level in circulation depends on different kinds of cytokine. This level also depends on different target cells used for gene transfer.

Keratinocyte gene therapy: inducible promoters and in vivo control of transgene expression

Modulation of transgene expression by exogenous agents is an optimal goal in gene therapy. Successful keratinocyte gene therapy requires a promoter–enhancer cassette to regulate expression of the therapeutic gene in vivo. In this study, we first transferred plasmids, constructed by introducing inducible promoters fused to the β‐galactosidase gene (LAC Z), into keratinocytes in vitro. Metallothionein (MT) and 1,24‐vitamin D3(OH)2 dehydroxylase (VDH) promoters responded to the inducing agents, Cadmium and 1,25‐vitamin D3(OH)2 (VitD3), respectively. The plasmids were then introduced in vivo using a naked DNA method and the inducible promoters were evaluated by measuring β‐gal activity in rat keratinocytes. Zinc induced the transferred MT promoter activity by ≈ 2‐fold or 10‐fold when administered systemically and topically, respectively. In addition, VitD3 induced the transferred VDH promoter activity ≈ 10‐fold when administered topically. These data are useful for developing inducible promoters for keratinocyte gene therapy.

A KERATIN K10 GENE MUTATION IN A JAPANESE PATIENT WITH EPIDERMOLYTIC HYPERKERATOSIS

SummaryEpidermolytic hyperkeratosis (EHK), or bullous congenital ichthyosiform erythroderma, is characterized by generalized erythroderma, ichthyosiform skin and blistering, and is caused by an aberration of the keratin intermediate filaments. In this study, we examined keratin K10 and 1 gene mutations in a Japanese EHK patient who had severe ichthyosiform erythroderma at birth and developed subsequent blistering. The patient had a G to A transition at codon 156 of the keratin K10 gene, which resulted in an arginine (Arg)→histidine (His) substitution in the helix initiation peptide of the highly-conserved 1A domain in keratin K10. This is the first mutation report of a Japanese patient with EHK, although the position and mode of the mutation identified here did not differ from those in reported Western cases.

Expression vector with DNA of bovine papilloma virus 1 for keratinocyte gene therapy.

Although there are several methods for introducing the genes to keratinocytes in vivo, expression of transgene does not last long enough for effective keratinocyte gene therapy. In this study, we added bovine papilloma virus 1 (BPV) DNA into expression vectors with the lacZ gene driven by metallothionein and keratin 10 promoters, and we transferred them into keratinocytes in vivo using the naked DNA method, and measured beta-gal activity in keratinocytes. The results showed that beta-galactosidase activity of vectors with the BPV DNA was clearly higher than that without the DNA. Moreover, time-course experiment disclosed that the activity of the BPV vector declined at a lower rate than that of the control vector, suggesting this fragment prolonged transgene expression. These results should prove useful for understanding gene regulation in keratinocytes in vivo and for developing potential expression vectors for keratinocyte gene therapy.

In Vivo Gene Transfer Method in Keratinocyte Gene Therapy: Intradermal Injection of DNA Complexed with High Mobility Group-1 Protein in Rats

In order to develop a more efficient method of introducing genes into keratinocytes in vivo, we intradermally injected DNA bound to high mobility group 1 protein, thereby taking advantages of the naked DNA and hemagglutinating virus of the Japan-liposome method reported previously. First we performed a gel mobility shift assay, which confirmed DNA binding to high mobility group 1. Then we injected beta-galactosidase expression vector complexed with high mobility group 1 into the rat skin and the activity of sample with the protein was 2-3 times higher than that without the protein as control. Semiquantification of transferred-DNA content using polymerase chain reaction and a time course of transgene expression in keratinocytes suggested that high mobility group 1 protein increased transfer of the DNA from the cytoplasm to the nucleus. Direct injection of the DNA-high-mobility-group-1 complex is a highly efficient method for introducing genes into keratinocytes in connection with gene therapy.