Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

Abstract

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of antiadenovirus immune responses expression in the brain lasts 6-18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1 × 10 7 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 × 10 7 -1 × 10³ iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8 + T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 × 10 7 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression.

In Vivo Polarization of IFN-γ at Kupfer and Non-Kupfer Immunological Synapses during the Clearance of Virally Infected Brain Cells

“…Coronal brain sections (50 m) were cut serially through the striatum, and immunofluorescence detection was performed as described (1,26), using the following primary Abs recognizing: TK (1:10,000; rabbit polyclonal, custom made by our laboratory) (1,25), TK (1:1,000; chicken polyclonal, custom made by AvesLabs Inc.) (27), LFA-1 (1:500; mouse monoclonal, IgG2a, BD PharMingen, clone WT.1), TCR (1:100; mouse monoclonal anti-TCR␣␤, IgG1, BD PharMingen, clone R73), IFN-␥ (1:100; goat polyclonal anti-rat IFN-␥, R&D systems), granzyme-B (prediluted; rabbit polyclonal Abcam), and macrophages/activated microglia (mouse anti-ED1;1: 1,000, Serotec, clone ED1).…”

Influence of phospholipid composition on cationic emulsions/DNA complexes: physicochemical properties, cytotoxicity, and transfection on Hep G2 cells

“…However, issues related to safety, risk of insertional mutagenesis, and the strong immune response generated by these vectors has limited its application. 2,4,5 Nonviral vectors have important safety advantages over viral approaches, including reduced pathogenicity and capacity for insertional mutagenesis, as well as low cost and ease of production. 6,7 However, application of non viral vectors has been held back by their poor efficiency in delivery to cells and transient expression of transgenes.…”

Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology

“…For example, the immune response to live influenza virus depends on the injection site (15); injection into the ventricles immediately causes immune priming, but injections into the brain parenchyma do not, until the infection breaks into the ventricles. Careful injections of bacillus Calmette-Guérin, a particulate nonsoluble antigen (16,17), or adenoviral vectors, directly into the brain parenchyma do not stimulate specific systemic immune responses, whereas injections into the ventricles do (18,19). Nevertheless, injections into the brain parenchyma will induce innate immune responses (e.g., cytokine release, TLR activation) restricted to the brain (20)(21)(22)(23).…”

“…Once the systemic immune system has been primed through peripheral exposure to antigen, effector lymphocytes have few restrictions in entering the brain and eliminating cells expressing their cognate antigens (24,25). The possibility therefore arises that if the population of DCs in the brain could be increased, immune surveillance of the brain would be improved.…”