Controlling therapeutic protein expression via inhalation of a butter flavor molecule

Abstract Precise control of the delivery of therapeutic proteins is critical for gene- and cell-based therapies, and expression should only be switched on in the presence of a specific trigger signal of appropriate magnitude. Focusing on the advantages of delivering the trigger by inhalation, we have developed a mammalian synthetic gene switch that enables regulation of transgene expression by exposure to the semi-volatile small molecule acetoin, a widely used, FDA-approved food flavor additive. The gene switch capitalizes on the bacterial regulatory protein AcoR fused to a mammalian transactivation domain, which binds to promoter regions with specific DNA sequences in the presence of acetoin and dose-dependently activates expression of downstream transgenes. Wild-type mice implanted with alginate-encapsulated cells transgenic for the acetoin gene switch showed a dose-dependent increase in blood levels of reporter protein in response to either administration of acetoin solution via oral gavage or longer exposure to acetoin aerosol generated by a commercial portable inhaler. Intake of typical acetoin-containing foods, such as butter, lychees and cheese, did not activate transgene expression. As a proof of concept, we show that blood glucose levels were normalized by acetoin aerosol inhalation in type-I diabetic mice implanted with acetoin-responsive insulin-producing cells. Delivery of trigger molecules using portable inhalers may facilitate regular administration of therapeutic proteins via next-generation cell-based therapies to treat chronic diseases for which frequent dosing is required.

: RNA-seq data of HEK293T cells untreated and treated with acetoin [10 mM]. For the 50 genes with top variance of expression, no clear clustering is seen on a heatmap of log2 of number of reads. Since overall fold changes and log2 of number of reads are low, there appears to be no marked difference between treated cells (T1-3) and untreated control (C1-3).
The RNAseq data presented in this figure have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible via GEO Series accession number GSE217626 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE217626).     NLuc over a period of 48 hours to monitor protein production capability over time as a control for the AIGES off-switching characteristics in Figure 3c. HEK-AIGES cells were seeded in a 6-well plate and induced for either 0, 8 16 or 24 hours with acetoin (10 mM). The medium was exchanged at timepoint 0 to fresh medium without acetoin, and reporter protein production was measured over 48 hours.  after induction, while the insulin levels decrease continuously during this period, returning to a normal fasting level 18 hours after induction.

Figure S8
: HEK-AIGESins cells were encapsulated and injected i.p. into diabetic mice. To assess long-term functionality, blood glucose levels were measured at 3 and 5 days after injection of the capsules. While blood glucose levels of the negative control and the uninduced group remained high, the induced group showed significantly reduced fasting glucose levels at 3 and 5 days after injection of the capsules. Mice were administered acetoin o.g. 16 hours prior to blood glucose measurements and were fasted overnight. Each group consists of 8 mice.      Mammalian SEAP reporter protein expression vector from the PYB-tata minimal promoter.
This work pAB003 AcoR from Bacillus subtilis expression plasmid PmPGK was restricted using MluI and EcoRI from pMM328 and inserted into the corresponding sites of pAB002 This work pAB004 AcoR from Bacillus subtilis expression plasmid PSV40 was restricted using MluI and EcoRI from SEAP2-Control and inserted into the corresponding sites of pAB002 (PSV40-VP16f-type-AcoR-pAbGH).
This work pAB009 AcoR from Bacillus subtilis expression plasmid This work VP16f-type was restricted using SpeI and HindIII from pTS2366 and inserted into NheI HindIII restricted pAB001.
This work pAB014 AcoR from Bacillus subtilis expression plasmid PhEF1a was restricted using MluI and EcoRI from pVH21 and inserted into the corresponding sites of pAB002 (PhEF1a-VP16f-type-AcoR-pAbGH).
This work pAB015 AcoR from Bacillus subtilis expression plasmid PmPGK was restricted using MluI and EcoRI from pMM328 and inserted into the corresponding sites of pAB009. (PmPGK-AcoR-VP16f-type-pAbGH).
This work pAB018 AcoR from Bacillus Pumilus containing FLAG sequence expression plasmid This work FLAG was obtained through annealing and phosphorylation of oAB121 (ctagtGGTGGTTCTGGTGACTACAAGGATGACGATGACAAGGCTAGCg) and oAB122 (gatccGCTAGCCTTGTCATCGTCATCCTTGTAGTCACCAGAACCACCa) and ligation into NheI BamHI restricted pAB006.
This work pAB108 AcoR-inducible mammalian SEAP reporter protein expression vector with consensus sequence 1,2' PhCMVmin SEAP reporter expression vector OAcoR was obtained through annealing and phosphorylation of oAB017 (cGAGACAAAATgcatgaTAgcatgaATTTTGTCTCa) and oAB018 This work (tcgatGAGACAAAATtcatgcTAtcatgcATTTTGTCTCggtac) and ligation into KpnI XhoI restricted pTS1015.
This work pAB111 AcoR-inducible mammalian SEAP reporter protein expression vector with consensus sequence 1,2' PhCMVmin SEAP reporter expression vector OAcoR was obtained through annealing and phosphorylation of oAB023 (cgcatgaAAATGAGACATAgcatgaATTTTGTCTCa) and oAB024 This work (tcgatGAGACAAAATtcatgcTATGTCTCATTTtcatgcggtac) and ligation into KpnI XhoI restricted pTS1015.
OAcoR was obtained through annealing and phosphorylation of oAB025 (c gcgtggtaccctcgagactcttcatacgttgGAGACAAAATGAGACATATGTCTCATTTTGTCTCa) and This work oAB026 (tcgatGAGACAAAATGAGACATATGTCTCATTTTGTCTCcaacgtatgaagagtctcgagggtacca) and ligation into MluI XhoI restricted pAB111.
This work pAB800 Stable Sleeping Beauty integration plasmid bearing AcoR and a blasticidin resistance gene.
PAcoO-PCMVmin-SEAP was restricted using MluI and NheI form pAB101 and mCherrymFT was restricted using SpeI and BamHI from mCherrymFT. Both were sequentially inserted into pTS1214 restricted with MluI SpeI and NheI BamHI to build the intermediate product pAB051 (PAcoO-PCMVmin-SEAP-p2a-This work mCherrymFT-pAbGH). pAB051 was then restricted using MluI and HindIII and inserted in the corresponding site of pTS2341.
OAcoR-PhCMVmin was restricted using MluI and EcoRI from pAB101 and inserted into the corresponding sites of pBS828.