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A green tea–triggered genetic control system for treating diabetes in mice and monkeys

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Science Translational Medicine  23 Oct 2019:
Vol. 11, Issue 515, eaav8826
DOI: 10.1126/scitranslmed.aav8826
  • Fig. 1 Design and validation of a PCA-inducible switch (PCAON) in mammalian cells and mice.

    (A) Summary of the core design and multiple use cases for the PCAON gene switch. (B) Detailed schematic for the PCAON switch design. The synthetic mammalian PCA-triggered transrepressor PcaR (KRAB-PcaV) is an N-terminal fusion of PcaV with a trans-silencing Krueppel-associated box (KRAB) domain: In the absence of PCA, PcaR binds to a chimeric target promoter PPcaR7 and represses SEAP expression; in the presence of PCA, PcaR is released from PPcaR7 and initiates SEAP expression. (C) PCA-inducible SEAP expression in different mammalian cell lines cotransfected with pJY14 (PPcaR7-SEAP-pA) and pJY29 (PhEF1α-PcaR-pA) was cultivated for 48 hours in the presence or absence of PCA. (D) Dose-dependent PCA-inducible SEAP expression. pJY14/pJY29-transgenic HEK-293 cells were cultivated with different concentrations of PCA. Different color bars represent different time periods for profiling SEAP expression. (E) Selection of stable PCAON transgenic cell lines (HEKPCA-ON-SEAP) was integrated with pJY14, pJY29, and pJY60 (PhCMV-PuroR-pA). The selected cell clones were profiled for their PCA-inducible SEAP regulation performance. (F) Reversibility of HEKPCA-ON-SEAP-mediated SEAP expression. HEKPCA-ON-SEAP cells (5 × 104) were cultivated for 6 days while alternating the PCA concentrations from 0 to 500 μM, and SEAP expression in the culture supernatants was profiled every 12 hours. Cell density was adjusted to 5 × 104 every 2 days. (G) Dose-dependent SEAP expression in HEKPCA-ON-SEAP cells. (H) Long-term PCAON-dependent SEAP expression. (I to K) PCA-dependent SEAP expression in mice. Mice implanted with microencapsulated HEKPCA-ON-SEAP cells received intraperitoneal (I) or oral (J) administration of PCA three times per day (0 to 500 mg kg−1 day−1) or drank different volumes of the concentrated green tea (K) (0 to 900 μl mouse−1 day−1, equivalent to 0 to 6.3 mg mouse−1 day−1 of total tea polyphenols); SEAP expression in bloodstream was profiled at 48 hours after implantation. The data in (C) to (H) represent the mean ± SD; n = 3 independent experiments. The animal data in (I) to (K) represent the mean ± SEM; two-tailed Student’s t test, n = 6 mice. n.s., not significant. All individual-level data are in data file S2.

  • Fig. 2 PCA-controlled CRISPR-Cas9 devices for genome and epigenome editing.

    (A) Schematic design of PCA-controlled PcaR-mediated inhibition (PcaRi). In the presence of PCA, the expression of both gRNA and dCas9-KRAB is induced to assemble into a repressive complex (gRNA-dCas9-KRAB) that targets a specific DNA site to inhibit gene expression. (B) Dose-dependent PCA-repressible SEAP expression for PcaRi. HEK-293 cells were cotransfected with pJY19 (PCAG-PcaR-pA), pJY131 (PPcaR12-dCas9-KRAB-pA), pJY109 (PgRNA1-SEAP-pA), and pJY53 (PPcaR13-gRNAASCL1) and cultivated with various PCA concentrations. SEAP expression was profiled after 48 hours. (C and D) PcaRi-mediated inhibition of endogenous gene expression. HEK-293 cells were cotransfected with pJY19, pJY131, pWL67 (PPcaR13-gRNACXCR4) (C), or pWL66 (PPcaR13-gRNATP53) (D) and cultivated for 48 hours with various PCA concentrations. The relative mRNA expression of CXCR4 (C) and TP53 (D) was quantified by qPCR. (E) Schematic design of PCA-controlled PcaR-mediated activation (PcaRa). In the presence of PCA, gRNAMS2 (gRNA with MS2 loop) and the transactivator MS2-p65-HSF1 are produced to recruit constitutively expressed dCas9 to form a transcriptional activation complex (gRNAMS2-dCas9-MS2-p65-HSF1). (F) Dose-dependent PCA-inducible SEAP expression for PcaRa. HEK-293 cells were cotransfected with pJY19, pSZ69 (PhCMV-dCas9-pA), pJY137 (PPcaR2-MS2-p65-HSF1-P2A-EGFP-pA), pJY110 (PgRNA2-SEAP-pA), and pJY57 [PPcaR14-gRNAASCL1(MS2)] and cultivated with various PCA concentrations. SEAP expression was profiled after 48 hours. (G and H) PcaRa-mediated activation of endogenous gene expression. HEK-293 cells were cotransfected with pJY19, pSZ69, pJY137, and pJY57 (G) or pJY55 [PPcaR14-gRNAPDX1(MS2)] (H) and cultivated for 48 hours with various PCA concentrations. The relative mRNA expression of ASCL1 (G) and PDX1 (H) was quantified by qPCR. (I) Schematic design of PCA-controlled PcaR-mediated gene deletion (PcaRdel). gRNA expression is induced by PCA and enables Cas9-mediated target gene deletion. (J) Correction of frameshift EGFP (fsEGFP) expression with PcaRdel. HEK-293 cells were cotransfected with pJY19 (PCAG-PcaR-pA), pJY58 (PPcaR14-gRNACCR5), pYW54 (PhCMV-Cas9-pA), and pJY221 (PhCMV-fsEGFP-pA) and cultivated in the presence or absence of 500 μM PCA. EGFP expression was profiled by fluorescence microscopy and flow cytometric analysis after 24 hours. The data (B to D, F to H, and J) represent the mean ± SD; n = 3 independent experiments. (K and L) PcaRdel-mediated genome editing. HEK-293 cells were cotransfected with pJY19 and pYW54, as well as pJY58 (K) or pJY59 (PPcaR14-gRNAEMX1) (L) and cultivated for 48 hours in the presence or absence of 500 μM PCA. Control cells were cotransfected with a constitutive Cas9 expression vector, as well as mismatched or matched gRNAs as negative or positive controls, respectively. Indel mutation frequencies of the CCR5 (K) and EMX1 (L) loci were evaluated using mismatch-sensitive T7E1 assays (n = 1 from two independent experiments). Black arrows indicate the expected cleavage bands. N.D., not detectable. All individual-level data are in data file S2.

  • Fig. 3 PCA- and VA-controlled programmable biocomputers in mice.

    The processing performance of the (A) A NIMPLY B, (B) B NIMPLY A, (C) AND, (D) OR, and (E) NOR logic gates in the implanted biocomputer in mice. Mice were intraperitoneally implanted with 2 × 106 microencapsulated (A) pJY29-/pJY162-/pJY179, (B) pJY12-/pJY159-/pCK189, (C) pJY29-/pCK189-/pJY303, (D) pJY29-/pDL24-/pCK189-/pDL30-/pDL6, and (E) pJY12-/pJY201-/pJY179-/pJY200-/pMF111 transgenic HEK-293 cells; these mice received thrice daily injections of different combinations of the two input signals PCA (500 mg kg−1 day−1) and VA (500 mg kg−1 day−1) in accordance with the truth table. The SEAP expression in the bloodstream was profiled at 48 hours after implantation. All data represent the mean ± SEM; two-tailed Student’s t test, n = 5 mice. See table S3 for detailed description of genetic components and table S6 for detailed transfection mixtures for each logic gate. All individual-level data are in data file S2.

  • Fig. 4 PCAON-1.0 switch-controlled treatment of type 1 and type 2 diabetic mice.

    (A) Long-term PCAON-regulated SEAP expression in mice. Mice implanted with 2 × 106 microencapsulated HEKPCA-ON-SEAP cells (200 cells per capsule) received thrice daily injections of PCA (500 mg kg−1 day−1) or oral administration of concentrated green tea (900 μl mouse−1 day−1). SEAP expression in the bloodstream of mice was monitored for 15 days. (B to D) PCA-dependent treatment of type 1 diabetic mice. Type 1 diabetic mice were intraperitoneally implanted with 3 × 106 microencapsulated HEKPCA-ON-SEAP-P2A-mINS cells and received thrice daily PCA injections (500 mg kg−1 day−1). Control mice were implanted with negative-control implants (HEKPCA-ON-SEAP cells) and received PCA injections, or mice received therapeutic implants (HEKPCA-ON-SEAP-P2A-mINS cells) but without PCA administration. (B) Blood insulin and (C) blood glucose were profiled for up to 15 days after implantation. (D) Intraperitoneal glucose tolerance test (IPGTT) was conducted on day 5 after implantation. (E to I) PCA-dependent treatment of type 2 diabetic mice. Type 2 diabetic mice were intraperitoneally implanted with 3 × 106 microencapsulated HEKPCA-ON-shGLP-1-P2A-SEAP cells and received thrice daily PCA injections (500 mg kg−1 day−1). Control mice were implanted with negative-control implants (HEKPCA-ON-SEAP cells) and received PCA injections, or mice received therapeutic implants (HEKPCA-ON-shGLP-1-P2A-SEAP cells) but without PCA administration. Blood (E) shGLP-1 and (F) glucose were quantified for up to 15 days after implantation. (G) IPGTT and (H) insulin tolerance test (ITT) were conducted, respectively, on days 5 and 7 after implantation. (I) Homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed on day 12 after implantation. Pink area represents normal blood glucose range. All data represent the mean ± SEM; two-tailed Student’s t test, n = 5 or 6 mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. All individual-level data are in data file S2.

  • Fig. 5 Design and validation of the PCAON switch 2.0 version (PCAON-2.0) in mammalian cells and mice.

    (A) Detailed schematic for the PCAON-2.0 switch design. On the basis of the PCAON-1.0 switch, the enhanced version PCAON-2.0 was designed based on pumping more PCA into engineered cells by a PCA transporter. In the presence of the transporter PcaK, PCA molecules were easily pumped from culture medium into cells, which results in the release of PcaR from PPcaR7 and the initiation of SEAP expression in a relative low PCA concentration. (B) Dose-dependent SEAP expression kinetics of the PCAON-2.0 switch compared with PCAON-1.0 switch. HEK-293 cells were transfected with either PCAON-1.0 switch (pJY29/pJY14) or PCAON-2.0 switch (pJY29/pJY14/pJY322) and cultivated with different concentrations of PCA. SEAP expression was profiled after 48 hours. (C) Time-dependent SEAP expression kinetics of the PCAON-2.0 switch compared with PCAON-1.0 switch. HEK-293 cells were transfected with either PCAON-1.0 switch (pJY29/pJY14) or PCAON-2.0 switch (pJY29/pJY14/pJY322) and cultivated for 72 hours in the presence or absence of 50 μM PCA. (D and E) The sensitivity comparison between PCAON-1.0- and PCAON-2.0-mediated SEAP expression folds in stable cell lines. (D) HEK-293 cells transgenic for PCAON-1.0- or PCAON-2.0- inducible SEAP and mouse insulin expression (HEKPCA-1.0-SEAP-P2A-mlINS or HEKPCA-2.0-SEAP-P2A-mlINS) were cultivated in medium containing various PCA concentrations. SEAP expression in the culture supernatants was profiled after 48 hours. (E) HEK-293 cells transgenic for PCAON-1.0- or PCAON-2.0- inducible SEAP and shGLP-1 expression (HEKPCA-1.0-shGLP-1-P2A-SEAP or HEKPCA-2.0-shGLP-1-P2A-SEAP) were cultivated in medium-containing various PCA concentrations. SEAP expression in the culture supernatants was profiled after 48 hours. (F to H) PCAON-2.0- dependent SEAP expression in wild-type mice. Mice implanted with microencapsulated HEKPCA-ON-2.0-shGLP-1-P2A -SEAP cells received intraperitoneal (F) or oral (G) administration of PCA three times per day (0 to 500 mg kg−1 day−1) or drank different volumes of the concentrated green tea (H) (0 to 900 μl mouse−1 day−1); SEAP expression in bloodstream was profiled at 48 hours after implantation. The data in (B) to (E) represent the mean ± SD; n = 3 independent experiments. The animal data in (F) to (H) represent the mean ± SEM; two-tailed Student’s t test, n = 6 mice. All individual-level data are in data file S2.

  • Fig. 6 PCAON-2.0 switch–controlled treatment of type 1 and type 2 diabetic mice achieved with orally infused PCA or tea drinking.

    (A to C) PCAON-2.0 switch–mediated treatment of type 1 diabetic mice. Type 1 diabetic mice implanted with 4 × 106 microencapsulated HEKPCA-2.0-SEAP-P2A-mINS cells (200 cells per capsule) received oral administration of concentrated green tea (900 μl mouse−1 day−1) or PCA (500 mg kg−1 day−1) thrice daily. Control mice were treated with water (900 μl mouse−1 day−1). (A) Blood insulin and (B) blood glucose were profiled for 15 days after implantation. (C) Intraperitoneal glucose tolerance test was conducted on day 15 after implantation. (D to H) PCAON-2.0 switch–mediated treatment of type 2 diabetic mice. Type 2 diabetic mice implanted with 4 × 106 microencapsulated HEKPCA-2.0-shGLP-1-SEAP cells (200 cells per capsule) received oral administration of the concentrated green tea (900 μl mouse−1 day−1) or PCA (500 mg kg−1 day−1) thrice daily. Control mice were treated with water (900 μl mouse−1 day−1). (D) Blood shGLP-1 and (E) blood glucose were profiled for 15 days after implantation. (F) Intraperitoneal glucose tolerance test and (G) insulin tolerance test were conducted on days 16 and 15, respectively, after implantation. (H) Insulin resistance was analyzed on day 12 after implantation. Pink area represents normal blood glucose range. All data represent the mean ± SEM; two-tailed Student’s t test, n = 5 mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. All individual-level data are in data file S2.

  • Fig. 7 PCAON-1.0 switch–controlled treatment of type 1 diabetic NHPs.

    (A) Schematic of PCA-controlled cell-based therapy in monkeys. After administration of PCA, implanted microcapsules containing PCA-controlled engineered cells produce secreted protein therapeutics into the bloodstream of cynomolgus monkeys. (B) Validation of the PCAON system in monkeys. Cynomolgus monkeys were intraperitoneally implanted with 1.5 × 108 microencapsulated HEK-293PCA-ON-SEAP-P2A-mINS cells (200 cells per capsule) and received thrice daily PCA injections (150 mg kg−1 day−1). The control monkey received PBS instead of PCA. SEAP expression in the bloodstream of monkeys was profiled at 48 hours after implantation. SEAP expression of individual monkeys was represented as dot plots. (C to F) PCA-dependent treatment of type 1 diabetic monkeys. Two type 1 diabetic monkeys (no. 1 and no. 2) were intraperitoneally implanted with 1.5 × 108 microencapsulated HEKPCA-ON-SEAP-P2A-mINS cells and received thrice daily PCA injections (150 mg kg−1 day−1). (C) Blood insulin and (D) glucose were profiled over a 4-day preimplantation and 15-day postimplantation period. N.D., not detectable. Red arrows represent the time of microcapsule implantation. Pink area represents normal blood glucose range. (E and F) Intravenous glucose tolerance of no. 1 and no. 2 monkeys was analyzed on day 5 after implantation. All individual-level data are in data file S2.

  • Fig. 8 PCAON-2.0 switch–controlled treatment of type 2 diabetic NHPs achieved with oral administration of PCA.

    (A) Schematic of PCAON-2.0 switch–controlled cell-based therapy in type 2 diabetic monkeys. After administration of PCA, implanted microcapsules containing PCAON-2.0 switch–controlled engineered cells produce shGLP-1 into the bloodstream of cynomolgus monkeys. (B to F) PCA-dependent treatment of type 2 diabetic monkeys. Two type 2 diabetic monkeys were intraperitoneally implanted with 2.5 × 108 microencapsulated HEKPCA-2.0-shGLP-1-P2A-SEAP cells and oral administration of thrice daily PCA solution (150 mg kg−1 day−1). (B) Blood shGLP-1 and (C) glucose were profiled over a 4-day preimplantation and 15-day postimplantation period. Red arrows represent the time of microcapsule implantation. Pink area represents normal blood glucose range. (D and E) Intravenous glucose tolerance of no. 1 and no. 2 monkeys was analyzed on day 5 after implantation. (F) Insulin resistance was analyzed on day 12 after implantation. All individual-level data are in data file S2.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/515/eaav8826/DC1

    Materials and Methods

    Fig. S1. Assessment of PCA-mediated toxicity on HEK-293 cells.

    Fig. S2. Optimization of the PCAON system in HEK-293 cells.

    Fig. S3. PCA analog-mediated SEAP expression in pJY14/pJY29-transgenic HEK-293 cells.

    Fig. S4. PCAON-dependent SEAP expression kinetics in HEK-293 cells.

    Fig. S5. Design, construction, and optimization of the PcaR-mediated inhibition device (PcaRi) for gene inhibition.

    Fig. S6. Design, construction, and optimization of the PcaR-mediated activation device (PcaRa) for gene activation.

    Fig. S7. Schematics of a synthetic PcaR-mediated gene deletion device (PcaRdel).

    Fig. S8. Controls with constitutively active CRISPR-dCas9 device-mediated genome repression and activation.

    Fig. S9. PCA- and VA-controlled programmable biocomputers in mammalian cells.

    Fig. S10. Flow cytometric histograms showing input-triggered single-cell d2EYFP expression of all programmed logic circuits.

    Fig. S11. Validation of the VAON and the VAOFF system in mammalian cells.

    Fig. S12. Validation of the PCAOFF system in mammalian cells.

    Fig. S13. Construction and characterization of the stable cell lines.

    Fig. S14. PCAON-2.0 switch-controlled treatment in type 1 diabetic mice by oral delivery of PCA.

    Fig. S15. Hypoglycemic effect on type 1 and type 2 diabetic mice by oral administration of PCA or tea drinking.

    Table S1. The CBC and blood biochemistry tests in type 1 diabetic monkeys.

    Table S2. The CBC and blood biochemistry tests in type 2 diabetic monkeys.

    Table S3. Plasmids designed and used in this study.

    Table S4. The primers used for qPCR analysis.

    Table S5. The primers used for PCR amplification.

    Table S6. The expression vectors and mixtures for logic gates in mice.

    Table S7. The expression vectors and mixtures for logic gates in mammalian cells.

    Data file S1. DNA sequence information of plasmids used in this study.

    Data file S2. Individual subject-level data.

    References (5364)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Assessment of PCA-mediated toxicity on HEK-293 cells.
    • Fig. S2. Optimization of the PCAON system in HEK-293 cells.
    • Fig. S3. PCA analog-mediated SEAP expression in pJY14/pJY29-transgenic HEK-293 cells.
    • Fig. S4. PCAON-dependent SEAP expression kinetics in HEK-293 cells.
    • Fig. S5. Design, construction, and optimization of the PcaR-mediated inhibition device (PcaRi) for gene inhibition.
    • Fig. S6. Design, construction, and optimization of the PcaR-mediated activation device (PcaRa) for gene activation.
    • Fig. S7. Schematics of a synthetic PcaR-mediated gene deletion device (PcaRdel).
    • Fig. S8. Controls with constitutively active CRISPR-dCas9 device-mediated genome repression and activation.
    • Fig. S9. PCA- and VA-controlled programmable biocomputers in mammalian cells.
    • Fig. S10. Flow cytometric histograms showing input-triggered single-cell d2EYFP expression of all programmed logic circuits.
    • Fig. S11. Validation of the VAON and the VAOFF system in mammalian cells.
    • Fig. S12. Validation of the PCAOFF system in mammalian cells.
    • Fig. S13. Construction and characterization of the stable cell lines.
    • Fig. S14. PCAON-2.0 switch-controlled treatment in type 1 diabetic mice by oral delivery of PCA.
    • Fig. S15. Hypoglycemic effect on type 1 and type 2 diabetic mice by oral administration of PCA or tea drinking.
    • Table S1. The CBC and blood biochemistry tests in type 1 diabetic monkeys.
    • Table S2. The CBC and blood biochemistry tests in type 2 diabetic monkeys.
    • Table S3. Plasmids designed and used in this study.
    • Table S4. The primers used for qPCR analysis.
    • Table S5. The primers used for PCR amplification.
    • Table S6. The expression vectors and mixtures for logic gates in mice.
    • Table S7. The expression vectors and mixtures for logic gates in mammalian cells.
    • References (5364)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). DNA sequence information of plasmids used in this study.
    • Data file S2 (Microsoft Excel format). Individual subject-level data.

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