An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1 beta enhancer region (-3690 to -2720) upstream of the human IL-6 promoter region (-163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (10(6) ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6(-/-) and IL-1(-/-) gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.

OBJECTIVE: To achieve disease-inducible expression of recombinant antiinflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. METHODS: We compared the inducible 2-component expression system (C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV]) with the constitutive cytomegalovirus (CMV) promoter in the polyarticular collagen-induced arthritis (CIA) model in mice. DBA/1 mice were immunized with bovine type II collagen and were given boosters on day 21. On day 22, mice were injected intraarticularly with the adenoviral vectors AdCMVLuc, AdCMVhIL-1Ra, AdC3-Tat/HIV-Luc, or AdC3-Tat/HIV-hIL-1Ra. The injected knee joints and hind paws were then scored for signs of arthritis, and knee joint histology was compared. RESULTS: The CMV-driven interleukin-1 receptor antagonist (IL-1Ra) expression resulted in a high constitutive expression and amelioration of CIA. C3-Tat/HIV-driven IL-1Ra expression could be detected only on days 24, 29, and 35. Fourteen days after injection of the vectors, CIA was significantly better inhibited by the C3-Tat/HIV-driven IL-1Ra expression compared with the CMV-driven IL-1Ra expression. Moreover, prevention of CIA in the knee joints also prevented CIA in the untreated hind paws. CONCLUSION: Our data demonstrate for the first time the feasibility of an inducible expression system for local production of IL-1Ra for treatment of arthritis in the CIA model.

Endogenous regulation of a therapeutic transgene restores homeostasis in arthritic joints.

The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-alpha, IL-1 beta, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall-induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.

In vitro evaluation of an enhanced human serum amyloid A (SAA2) promoter-regulated soluble TNF receptor fusion protein for anti-inflammatory gene therapy.

Tumour necrosis factor (TNF)-alpha contributes to the pathogenesis of many inflammatory diseases. Recombinant soluble TNF receptor fusion proteins (sTNFR:Ig) are potent TNF antagonists, both in vitro and in vivo. The concentration of serum amyloid A (SAA) increases by up to 1000-fold during inflammation, largely owing to cytokine-driven transcriptional upregulation. A reporter plasmid, comprising the proximal 0.7 kb of the human SAA2 promoter fused to a luciferase gene, was used in transient transfection experiments in human HepG2 hepatoma cells to assess the quantitative and qualitative TNF antagonist properties of a construct in which sTNFR:Ig synthesis is under the control of a chimera of the SAA2 promoter and a tat/HIV element. The SAA2-tat/HIV-sTNFR:Ig construct retained the fine-tuned cytokine responsiveness of the SAA2 promoter, while exhibiting the quantitatively enhanced level of protein expression conferred by the tat/HIV element. It produced a biologically significant TNF inhibition that was at least as strong as that achieved using a CMV promoter-driven sTNFR:Ig construct. There was a dose- and time-dependent relationship between the pro-inflammatory cytokine used, and the generation of TNF antagonist activity by SAA2-tat/HIV-sTNFR:Ig. Although sTNFR:Ig protein can be induced by either TNF-alpha or interleukin (IL)-1beta, its antagonist activity is limited to the former cytokine. The SAA2-tat/HIV-sTNFR:Ig construct, and derivatives thereof, may therefore be ideally suited to gene therapy applications that require the local production of potent and specific immune modifiers only when there is active pathology. It may consequently be of particular use in the future treatment of diseases such as rheumatoid arthritis.

Gene therapy to prevent organophosphate intoxication.

The specific hydrolytic activity of PON1 paraoxonase/arylesterase enzymes in liver and blood provides a natural barrier against the entry of organophosphate toxins into the central and peripheral nervous systems. Inherited differences in PON1 enzyme concentrations may determine levels of susceptibility to organophosphate injury in humans. To test whether boosting serum levels of PON1 enzymes by gene therapy might provide increased protection, we compared the degree of inactivation of whole brain acetylcholinesterase of mice exposed to chlorpyrifos 4 days after intravenous injection of recombinant adenoviruses containing PON1-LQ or PON1-LR genes or no PON1 gene. Both recombinant viruses containing PON1 genes boosted serum arylesterase concentrations by approximately 60% and significantly prevented the inactivation of brain acetylcholinesterase. Some mice were completely protected. These findings indicate that boosting serum levels of PON1 enzymes by a gene delivery vector raises the threshold for organophosphate toxicity by hydrolytic destruction before the chemical can enter the brain.

Physiologically responsive gene therapy.

The goal of physiologically responsive gene therapy is to allow a host's endogenous regulatory mechanisms to control the production of therapeutic proteins (effectors). Ideally, effector production would be switched on in response to specific signals, stay within therapeutic limits and be switched off when no longer needed. In this way, the unwanted consequences of constitutive, high-level effector expression could be avoided. While recent studies have shown that transgenes can be regulated within animal hosts, they have also highlighted significant problems that require much further research.

A two-component expression system that responds to inflammatory stimuli in vivo.

A therapeutic dilemma often complicates the management of inflammatory diseases; the benefits gained from reducing inflammation must be balanced against the potentially harmful consequences of chronic immunosuppression. Gene therapy might address this dilemma by producing anti-inflammatory proteins in response to a patient's endogenous signals, so that recombinant drug production is linked to the intensity and duration of the inflammatory condition. To test this, we have developed inflammation-inducible systems for regulating recombinant protein production in vivo. We describe a two-component expression construct in which (1) the murine complement factor 3 (C3) promoter regulates production of the human immunodeficiency virus (HIV) transactivator of transcription (Tat), and (2) the Tat protein then stimulates protein expression from genes inserted downstream of the the HIV promoter. When incorporated into a nonreplicating adenovirus (Ad.C3-tat/HIV-luc) and studied in a murine model, the construct produces large amounts of recombinant protein in vivo in response to two different inflammatory stimuli.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

The divIVB region of the Bacillus subtilis chromosome encodes homologs of Escherichia coli septum placement (minCD) and cell shape (mreBCD) determinants.

Mutation of the divIVB locus in Bacillus subtilis causes frequent misplacement of the division septum, resulting in circular minicells, short rods, and filaments of various sizes. The divIVB1 mutant allele maps to a region of the chromosome also known to encode sporulation (spo0B, spoIVF, spoIIB) and cell shape (rodB) determinants. This study reports the cloning and sequence analysis of 4.4 kb of the B. subtilis chromosome encompassing the divIVB locus. This region contains five open reading frames (ORFs) arranged in two functionally distinct gene clusters (mre and min) and transcribed colinearly with the direction of replication. Although sequence analysis reveals potential promoters preceding each gene cluster, studies with integrational plasmids suggest that all five ORFs are part of a single transcription unit. The first gene cluster contains three ORFs (mreBCD) homologous to the mre genes of Escherichia coli. We show that rodB1 is allelic to mreD and identify the rodB1 mutation. The second gene cluster contains two ORFs (minCD) homologous to minC and minD of E. coli but lacks a minE homolog. We show that divIVB1 is allelic to minD and identify two mutations in the divIVB1 allele. Insertional inactivation of either minC or minD or the presence of the divIVB region on plasmids produces a severe minicell phenotype in wild-type cells. Moreover, E. coli cells carrying the divIVB region on a low-copy-number plasmid produce minicells, suggesting that a product of this locus may retain some function across species boundaries.