Abstract
The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.
Highlights
Gene therapy aims to modify pathological phenotypes and provide disease treatment by the introduction of transgenes via recombinant viral vectors or non-viral vectors.[1]
We treated the mice with a single low dose of 1 mg TMP by oral gavage (50 mg/kg in the mouse, a human equivalent dose of 4.07 mg/kg,[47] which is considered “lowdose” TMP48) to mimic one antibiotic dosage, in practicality, regulation of the dihydrofolate reductase (DHFR) destabilizing domain (DD) system in a gene therapy context would consist of long-term and frequent treatment using TMP
We performed qPCR experiments on mouse fecal samples collected at day 0, day 3, and day 7 to quantify the abundance of total bacteria and several representative gut bacteria, including Bacteroides (BACT), Enterobacteriaceae (ENTERO), the Eubacterium rectale/Clostridium coccoides (EREC) group, the Clostridium leptum (CLEPT) group, and the Lactobacillus/Enterococcus (LACT) group
Summary
Gene therapy aims to modify pathological phenotypes and provide disease treatment by the introduction of transgenes via recombinant viral vectors (e.g., recombinant adeno-associated virus [rAAV] or lentivirus) or non-viral vectors (naked DNA, nanoparticles, etc.).[1]. DDs are genetically engineered domains that are inherently unstable and rapidly ubiquitinated and degraded by the proteasome, unless the DD is stabilized by a small-molecule pharmacologic chaperone.[9] Use of the Escherichia coli (E. coli) dihydrofolate reductase (DHFR) DD is appealing, due to its stabilizing ligand, trimethoprim (TMP), an inexpensive and well-characterized compound that can cross both the blood-brain barrier (BBB)[10] and the blood-retinal barrier (BRB)[11] and that is highly specific for E. coli DHFR.[12] In the presence of TMP, fusion proteins containing DHFR DDs are readily expressed and resistant to proteasomal degradation, allowing for positive regulation.[10] The DHFR DD system is reversible in that TMP can be washed out in vitro and metabolized or excreted in vivo This generalized model system has been confirmed to be effective in controlling the abundance of numerous fusion proteins[13,14,15,16,17,18,19] in several tissues, including the brain[10,20,21] and the eye,[11,22] in a spatial, temporal, and dose-dependent manner
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