Production and sorting of transgenic, modified human parathyroid hormone in vivo in rat salivary glands.

Polarized salivary epithelial cells can sort secretory proteins towards either the basolateral or apical pole. Transgenic human parathyroid hormone (hPTH) exclusively sorts apically in rat submandibular glands. To help understand this specific process we modified the hPTH cDNA sequence and delivered the cDNAs to glands in vivo using adenoviral (Ad) vectors. The Ad vectors encoded: (1) the native form of hPTH (Ad.pre-pro-hPTH1-84), (2) the native sequence, but with the pro-segment deleted (Ad.pre-hPTH1-84), and (3) a sequence containing the pre-segment followed by the first 34 amino acids of hPTH (Ad.pre-hPTH1-34). hPTH production and sorting were studied after two days. All constructs were effectively transcribed in targeted glands. However, the pre-hPTH1-84 modification led to reduced hPTH secretion and production, while no immunoreactive hPTH resulted from pre-hPTH1-34 cDNA infusion. The pre-hPTH1-84 modification had no effect on apical sorting. These in vivo results show that the signal responsible for hPTH's apical sorting does not reside in the pro-segment and that deleting both the pro-segment and the carboxyl-terminal region severely impairs post-translational processing of hPTH.

In vivo secretion of the mouse immunoglobulin G Fc fragment from rat submandibular glands.

BACKGROUND: Salivary glands have been proposed as target organs for gene therapy. They secrete endogenous, as well as transgenic proteins, in a polarized manner. Transgene-encoded regulated pathway proteins primarily follow the regulated pathway in rat salivary glands and are secreted into saliva in an exocrine manner. Conversely, constitutive pathway proteins generally are secreted more basolaterally and thus follow the endocrine route. In the present study, we studied in vivo the sorting of the mouse immunoglobulin G2b Fc fragment, which is physiologically secreted via the constitutive pathway. METHODS: Adenoviral vectors encoding the Fc fragment and human growth hormone were delivered into rat and mouse submandibular glands in vivo to compare their serum-to-saliva distribution. We also compared the intracellular localization of the Fc fragment and growth hormone by confocal microscopy. RESULTS: We found that the Fc fragment was secreted almost entirely into the bloodstream from rat and mouse submandibular glands via a constitutive or constitutive-like pathway. This sorting behaviour is clearly different from that of transgenic human growth hormone, which is secreted in a regulated pathway, both in neuroendocrine cells and as a transgenic protein from salivary gland cells. We also found that simultaneously expressed human growth hormone and the mouse Fc fragment do not appear to influence each other's sorting behaviour. The Fc fragment showed a primarily basal localization, whereas growth hormone showed an apical localization, in rat submandibular gland acinar cells. CONCLUSIONS: The results obtained in the present study indicate that the mouse Fc fragment is a useful model protein for examining the basolateral versus apical secretory pathways employed by transgenic secretory proteins in salivary glands.

Stability and suitability for storage and distribution of Ad26.ZEBOV/MVA-BN®-Filo heterologous prime-boost Ebola vaccine.

The stability profile of a vaccine has important implications for storage, cold chain management and field deployment. The heterologous prime-boost Janssen Ebola vaccine regimen demonstrated an acceptable safety profile and durability of Ebola-specific immune responses in Phase I studies in healthy adults. Potency (infectious titre) of both components of the Ad26.ZEBOV/MVA-BN-Filo regimen were assessed using qPCR-based potency assay and flow cytometry during real-time and accelerated stability studies, conducted between -80 °C and 25 °C. Additionally, vaccine potency was assessed following agitation, temperature cycling, freeze-thawing and while in the injection system. Ad26.ZEBOV remained stable for 24 months when frozen and at 2-8 °C; MVA-BN-Filo remained stable for 24 months frozen and 12 months at 2-8 °C. Potency of both vaccines was maintained during temperature cycling, agitation and freeze-thawing. When exposed to high temperatures (up to 40 °C) in a syringe/needle both vaccines remained stable for at least 6 h. The vaccines are expected to maintain potency for 36 months when frozen (based on extrapolation of observed stability). The findings of this study indicate that the stability of the Ad26.ZEBOV/MVA-BN-Filo is likely suitable for field deployment in regions at risk of Ebola outbreaks, where cold chain maintenance is challenging owing to infrastructure and resource limitations.

Amelioration of arthritis by intraarticular dominant negative Ikk beta gene therapy using adeno-associated virus type 5.

Nuclear factor (NF)-kappaB is highly activated in the synovium of rheumatoid arthritis (RA) patients, and can induce transcription of many proinflammatory molecules. Phosphorylation of inhibitor of kappaB (IkappaB) proteins is an important step in NF-kappaB activation and under inflammatory conditions is regulated predominantly by IkappaB kinase (IKK)beta. Consequently, specific targeting of IKK beta in the joint, using gene therapy, presents a sophisticated treatment option for arthritis. In the present study we investigated the effect of inhibiting IKK beta in adjuvant arthritis (AA) in rats, using recombinant adeno-associated virus (rAAV)-mediated intraarticular gene therapy. For this purpose rAAV5 carrying the dominant negative IKK beta gene (AAV5.IKK beta dn) or control AAV5.eGFP was injected into the right ankle joint. Rats treated with AAV5.IKK beta dn in early arthritis exhibited significantly reduced paw swelling (p < 0.05). Immunohistochemical analysis of synovial tissue revealed reduced levels of interleukin (IL)-6 (p = 0.005) and tumor necrosis factor-alpha (TNF-alpha) (p = 0.03), whereas IL-10 levels were not affected. No significant effect was found on cartilage and bone destruction, or on matrix metalloproteinase-3 and tissue inhibitor of matrix metalloproteinase-1 expression. Injection of AAV5.IKK beta dn in the preclinical phase showed only a marginal effect on arthritis. Importantly, in this study we also demonstrate for the first time that our vector is capable of transducing human RA whole synovial tissue biopsies ex vivo, resulting in reduced IL-6 production after TNF-alpha stimulation (p = 0.03). In conclusion, we are the first to demonstrate that rAAV5 can be used to successfully deliver a therapeutic gene (IKK beta dn) to the synovium, resulting in reduced severity of inflammation in AA in vivo and proinflammatory cytokine production in human RA synovial tissue ex vivo. This translational research represents a crucial next step toward the development of gene therapy for application in humans.

Salivary epithelial cells: an unassuming target site for gene therapeutics.

Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer.

Gene therapy of salivary diseases.

For many years, our laboratory has been developing gene transfer approaches for salivary gland disorders that currently lack effective therapy. The purpose of this chapter is to describe key methods used in this developmental process. Specifically, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model.

A novel approach for gene therapy: engraftment of fibroblasts containing the artificial chromosome expression system at the site of inflammation.

BACKGROUND: Rheumatoid arthritis is characterized by inflammation of the synovial tissue. High systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene transfer might provide a more efficient delivery system for genes encoding therapeutic proteins. METHODS: The artificial chromosome expression system (ACE System) is a new non-integrating, non-viral gene expression system which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of proteins encoded by single or multiple genes over long periods of time. We are developing ex vivo gene therapy using murine artificial chromosomes containing a reporter gene (LacZ and red fluorescent protein (RFP)) for local delivery of genes in rats with adjuvant arthritis (AA). RESULTS: The delivery of the intact ACE System into rat fibroblast-like synoviocytes (FLS) and rat skin fibroblasts (RSF) was detected within 24 to 48 h post-transfection. After growing cells under selection, clones expressing LacZ and RFP were identified. Furthermore, we investigated the feasibility of local delivery of a reporter gene to the joints of rats with AA by ex vivo gene therapy. This resulted in engraftment of the injected cells in the synovial tissue microarchitecture and expression of the reporter gene. CONCLUSIONS: This work demonstrates the potential feasibility of treating arthritis and other inflammatory diseases using fibroblasts containing the ACE System as a non-viral vector for gene therapy.

CD97 neutralisation increases resistance to collagen-induced arthritis in mice.

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and alpha5beta1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.