Abstract
On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.
Highlights
Nicotinamide adenine dinucleotide (NAD+) is a key molecule in cellular metabolism and acts as an intermediate in several essential enzymatic reactions [1]
When murine T cells are exposed to micromolar levels of extracellular NAD+, ARTC2.2 catalyzes the ADP-ribosylation of exposed arginines in several cell surface protein targets, including the purinergic P2X7, a well described protein expressed by immune cells and involved in immune regulation [10, 11]
We demonstrated here the ability to durably block the activity of ARTC2.2 enzyme or of the P2X7 ion channel in vivo upon a single i.m. injection of the recombinant AAV vectors (rAAV) encoding a construct containing the ARTC2.2-blocking nanobody s+16a, or a construct containing the P2X7-blocking nanobody 13A7
Summary
Nicotinamide adenine dinucleotide (NAD+) is a key molecule in cellular metabolism and acts as an intermediate in several essential enzymatic reactions [1]. Mono-ADP ribosyl transferases (ART) represent a family of ectoenzymes that use extracellular NAD+ to catalyze posttranslational modification of cell surface proteins by the transfer of ADP-ribose to specific amino-acid residues [4, 5]. While ARTC2.1 is enzymatically inactive in the absence of reducing agents, ARTC2.2 is active in standard conditions and is able to ADP-ribosylate multiple cell-surface protein-targets when NAD+ is present in the extracellular space [7, 8]. When murine T cells are exposed to micromolar levels of extracellular NAD+, ARTC2.2 catalyzes the ADP-ribosylation of exposed arginines in several cell surface protein targets, including the purinergic P2X7, a well described protein expressed by immune cells and involved in immune regulation [10, 11]
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