Inhibition of established collagen-induced arthritis with a tumour necrosis factor-α inhibitor expressed from a self-contained doxycycline regulated plasmid

Tumor necrosis factor (TNF)-α is produced by cells of the immune system and is a key mediator in immune and inflammatory reactions. Through interaction with widely expressed receptors (TNF receptor 1 and TNF receptor 2), TNF-α is able to orchestrate the expression of a range of downstream proinflammatory molecules. Over the past decade novel biologics that inhibit TNF-α have been developed as extremely effective treatments for rheumatoid arthritis. Structurally, these biologics are antibodies, or TNF receptors on an antibody backbone that bind TNF-α directly and are delivered to patients by repeated injection. Gene therapy offers an improved approach to delivering biologics as a single administration of their encoding genetic material. In the present study we demonstrate the therapeutic effect of a small molecular weight dimeric TNF receptor 2 (dTNFR) constitutively expressed from plasmid DNA, delivered intramuscularly with electroporation, after disease onset in a collagen-induced arthritis model. Regulated promoters that enable the production of a transgene to be controlled are more suited to the application of gene therapy in the clinic. Regulated expression of dTNFR from the plasmid pGTRTT was also therapeutic in the mouse collagen-induced arthritis model when the inducer doxycycline was also administered, whereas no therapeutic effect was observed in the absence of doxycycline. The therapeutic effect of dTNFR expressed from a constitutive or regulated plasmid was dependent on the degree of disease activity at the time of DNA injection. The observations of this study are considered with regard to the disease model, the magnitude of gene regulation, and the path to clinical application

Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis

Inhibition of tumour necrosis factor (TNF)-alpha with biological molecules has proven an effective treatment for rheumatoid arthritis, achieving a 20% improvement in American College of Rheumatology score in up to 65% of patients. The main drawback to these and many other biological treatments has been their expense, which has precluded their widespread application. Biological molecules could alternatively be delivered by gene therapy as the encoding DNA. We have developed novel plasmid vectors termed pGTLMIK and pGTTMIK, from which luciferase and a dimeric TNF receptor II (dTNFR) are respectively expressed in a doxycycline (Dox)-regulated manner. Regulated expression of luciferase from the self-contained plasmid pGTLMIK was examined in vitro in a variety of cell lines and in vivo following intramuscular delivery with electroporation in DBA/1 mice. Dox-regulated expression of luciferase from pGTLMIK of approximately 1,000-fold was demonstrated in vitro, and efficient regulation was observed in vivo. The vector pGTTMIK encoding dTNFR was delivered by the same route with and without administration of Dox to mice with collagen-induced arthritis. When pGTTMIK was delivered after the onset of arthritis, progression of the disease in terms of both paw thickness and clinical score was inhibited when Dox was also administered. Vectors with similar regulation characteristics may be suitable for clinical application

Targeted delivery of anti-inflammatory therapy to rheumatoid tissue by fusion proteins containing an IL-4-linked synovial targeting peptide

We provide first-time evidence that the synovial endothelium-targeting peptide (SyETP) CKSTHDRLC successfully delivers conjugated IL-4 to human rheumatoid synovium transplanted into SCID mice. SyETP, previously isolated by in vivo phage display and shown to preferentially localize to synovial xenografts, was linked by recombinant technology to hIL-4 via an MMP-cleavable sequence. Both IL-4 and the MMP-cleavable sequence were shown to be functional. IL-4-SyETP augmented production of IL-1ra by synoviocytes stimulated with IL-1[beta] in a dose-dependent manner. In vivo imaging confirmed increased retention of SyETP-linked-IL-4 in synovial grafts which was enhanced by increasing number of copies (one to three) in the constructs. Strikingly, SyETP delivered bioactive IL-4 in vivo as demonstrated by increased pSTAT6 in synovial grafts. Thus, this study provides proof of concept for peptide-tissue-specific targeted immunotherapy in rheumatoid arthritis. This technology is potentially applicable to other biological therapies providing enhanced potency to inflammatory sites and reducing systemic toxicity

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a "cross-talk" between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus - thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass.Facultad de Ciencias Veterinaria

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a "cross-talk" between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus - thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass.Facultad de Ciencias Veterinaria

Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.Facultad de Ciencias Veterinaria

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a "cross-talk" between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus - thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass.Facultad de Ciencias Veterinaria

Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.Facultad de Ciencias Veterinaria