Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant


Constitutive mammalian mYPet-YcgR-mCyPet expression vector (P SV40 -mYPet-YcgR-mCyPet-pA). mYPet-YcgR-mCyPet was excised from pMFO6 using EcoRI/PstI and cloned into the corresponding sites (EcoRI/PstI) of pSBC-2. 

This work pKZY113

Constitutive mammalian DGCL expression vector (P SV40 -DGCL-pA). DGCL was excised from pMFO5 using EcoRI/PstI and cloned into the corresponding sites (EcoRI/PstI) of pSBC-2. 

This work pKZY119

Constitutive mammalian PDE yahA expression vector (P SV40 -PDE yahA -pA). PDE yahA was excised from pMSO2 using EcoRI/PstI and cloned into the corresponding sites (EcoRI/PstI) of pSBC-2. 

This work pKZY120

Constitutive mammalian PDE TBD1265 expression vector (P SV40 -PDE TBD1265 -pA). PDE TBD1265 was excised from pMFO1 using EcoRI/PstI and cloned into the corresponding sites (EcoRI/PstI) of pSBC-2. 

This work pKZY121

Constitutive mammalian DGCA CC3285 expression vector (P SV40 -DGCA CC3285 -pA). DGCA CC3285 was excised from pMFO2 using EcoRI/PstI and cloned into the corresponding sites (EcoRI/PstI) of pSBC-2. 
This work pSO1 P hIFN -driven SEAP expression vector (P hIFN -SEAP-pA). P hIFN was PCR-amplified from HEK-293 genomic DNA using oligonucleotides OSO1 (5'-aatgagctagcgaattcTCAGGTCGTTTGCTTTC-3') and OSO2 (5'atgaaagcttcATGTTGACAACACGAACAGTG-3') restricted with NheI/HindIII and cloned into the corresponding site (NheI/HindIII) of pSEAP2-control. 
This work pSO2 P IFN(AC+) -driven SEAP expression vector (P IFN(AC+) -SEAP-pA). The IRF3 operator sites of P hIFN were sequence optimized by PCR-based site-directed mutagenesis using the oligonucleotides OSO3 (5'-AaAGGgAAACTGAAAGGGAaAACTGAAAGTGGGAAATTCCTCTG-3') and OSO4 (5'-GTCATT TACATTTTAGTAGTTTC-3') and pSO1 as template. 
This work pSO3 P IFN(ACD+) -driven SEAP expression vector (P IFN(ACD+) -SEAP-pA). The IRF3 operator sites of P IFN(AC+) were space optimized by PCR-based site-directed mutagenesis using the oligonucleotides OSO5 (5'-AAAGGG AAACTGAAAGGGAAACTGAAAGTGGGAAATTCCTCTG -3') and OSO4 (5'-GTCATTTACATTTT AGTAGTTTC-3') and pSO2 as template. 

This work pSO4

Constitutive mammalian DGCL expression vector (P hCMV -DGCL-pA). DGCL was excised from pKZY113 using EcoRI/HindIII and cloned into the corresponding sites (EcoRI/HindIII) of pMM1. 

This work

YcgR-mCyPet, N-terminally His6-tagged c-di-GMP biosensor consisting of a central Salmonella typhimurium-derived diguanylate receptor domain (YcgR) flanked by yellow (mYPet) and cyan (mCYPet) fluorescent protein domains; ori SV40 , simian virus 40-derived origin of replication; PDE yahA , cyclic diguanylate (c-di-GMP)-degrading phosphodiesterase of Escherichia coli (ATCC 8937) (human codon-optimized PDE yahA , GenBank ID: KM591194 (9); PDE TBD1265 , cyclic diguanylate (c-di-GMP)-degrading phosphodiesterase of Thiobacillus denitrificans (ATCC 25259) (human codon-optimized PDE TBD1265 , GenBank ID: KM591195)(10); P hCMV , human cytomegalovirus immediate early promoter; P hCMVmin , minimal version of P hCMV ; P hEF1 , human elongation factor 1 alpha promoter; P hIFN , human interferon beta promoter (GenBank ID: KM591197); P IFN(AC+) , modified P hIFN containing sequence-optimized IRF3 operator sites (GenBank ID: KM591198) (11); P IFN(ACD+) , P IFN(AC+) containing space-optimized IRF3 operator sites (GenBank ID: KM591199) (25); Pol, HIV virion-associated polymerase; P SV40 , simian virus 40 promoter; P T7 , bacteriophage T7 promoter; RRE, HIV-1-derived Rev response element; SEAP, human placental secreted alkaline phosphatase; STING, mouse stimulator of interferon genes; TAT, HIV-1-derived transactivator of transcription; VSV-G, vesicular stomatitis virus protein G;  + , extended HIV-1-derived lentiviral packaging signal. 

Suplementary References


Supplementary Figure 4 |

 4 Analysis of potential interference of the orthogonal c-di-GMPbased second messenger signalling pathway with hIFN- production and paracrine hIFN- signalling. (a) The orthogonal c-di-GMP-based second messenger signalling pathway fails to induce endogenous hIFN- secretion in engineered HEK-293T. 5x10 4 HEK-293T cells were cotransfected with pSTING (P hCMV -STING-pA) and pSO3 (P IFN(ACD+) -SEAP-pA) as well as optionally with pZKY121 (P SV40 -DGCA CC3285 -pA) and SEAP as well as hIFN- levels were profiled in the culture supernatant after 48h. HEK-293T transfected with the constitutive hIFN- expression vector pWW512 (P hEF1 -hIFN--pA) were used as positive control. (b) Paracrine hIFN- has no impact on the orthogonal c-di-GMP-based second messenger signalling pathway. 5x10 4 HEK-293T cells were cotransfected with pSTING (P hCMV -STING-pA) as well as pSO3 (P IFN(ACD+) -SEAP-pA) and exposed to recombinant hIFN- for 48h before SEAP expression was profiled in the culture supernatant. The STINGdependent type I interferon inducer DMXXA was used as positive control. Data are mean ± SD; n=5, triplicate experiments. restriction endonuclease-specific sites are underlined in oligonucleotide sequences. Annealing base pairs contained in oligonucleotide sequences are shown in capital letters. Abbreviations: 3'LTR ΔU3 , HIV-1-derived enhancer-free 3'LTR variant; 5'LTR, HIV-1-derived 5' long terminal repeat; c-di-GMP, cyclic diguanosine monophosphate; cPPT, central polypurine tract; DGCA CC3285 , feedback-inhibited CC3285 diguanylate cyclase A of Caulobacter crescentus (ATCC 19089/CB15) (human codon-optimized DGCA CC3285 , GenBank ID: KM591193) (7); DGCL, Near infrared-light-activated diguanylate cyclase derived from Rhodobacter sphaeroides BphG1 (ATCC BAA-808; N-terminal PAS-GAF-PHY-GGDEF portion of BphG1 (Q8VRN4_RHOSH) and the catalytic diguanylate cyclase domain GGDEF which is photo-activated by it's cognate PAS-GAF-PHY phytochrome) (human codon-optimzed DGCL, GenBank ID: KM591196 (8); EGFP, enhanced GFP; EYFP, yellow-fluorescent GFP variant; GFP, enhanced green-fluorescent protein; GAG, HIV-derived core protein; hIFN-, human interferon beta; HIV, human immunodeficiency virus; IRF3, interferon regulatory factor 3; pA, polyadenylation signal; MCS, multiple cloning site; mYPet-pKZY81 




  


Supplementary Information

Supplementary Figure 1 | Impact of constitutive diguanylate cyclase expression on the viability and metabolic capacity of mammalian cells. 5x10 5 HEK-293T were cotransfected with the constitutive Caulobacter crescentus diguanylate cyclase A (DGCA CC3285 ) expression vector pZKY121 (P SV40 -DGCA CC3285 -pA) and the constitutive SEAP expression vector pSEAP2-control (P SV40 -SEAP-pA) and cell viability (a) as well as the SEAP production capacity (b) were profiled for up to 72h. Data are mean ± SD; n=5, triplicate experiments. 

Supplementary


Supplementary Table 1. Plasmids and oligonucleotides used and designed in this study Plasmid

Description and Cloning Strategy Reference or Source pcDNA3.1(+) 
Constitutive P hCMV -driven mammalian expression vector (P hCMV -MCS-pA). Invitrogen pCD/NL/BH* HIV-1-derived GAG/Pol/TAT-encoding helper plasmid (P hCMV -GAG-Pol-TAT-pA). 
(1) pET15b::mYPet-ycgR-mCyPet Consitutive prokaryotic expression vector encoding mYPet-YcgR-mCyPet (P T7 -mYPet-YcgR-mCyPet). 
( 
pGEM ® -T Easy Vector for cloning of PCR products. Promega pLTR-G Constitutive mammalian VSV-G expression vector (5'LTR-VSV-G-pA). 
(3) pMM1 
Constitutive pcDNA3.1(+)-derived expression vector with modified MCS (P hCMV -MCS-pA; MCS, EcoRI-ATG-SpeI-NheI-BamHI-STOP-XbaI-HindIII-FseI-pA). pcDNA3.1 was PCR-amplified using oligonucleotides OMM1 (5-cggatccaccggtgtctagaaagctttgaggccggcctgcaggGATCAGCCTCGACTGTGCC TTC-3') and OMM2 (5'-ctagcactagtcatggtgaattcgattaatcaattgacgcgtGGAGATCTCCCGATCCGTC-3') and self-ligated. 

Mueller et al., unpublished pNLK8

HIV-1-derived constitutive lentiviral EYFP expression vector (5'LTR- + -ori SV40 -cPPT-RRE-P hEF1 -EYFP-3'LTR ΔU3 ). 
pSBC-2 Constitutive mammalian expression vector (P SV40 -MCS-pA). 
(5) pSEAP2-control Constitutive mammalian SEAP expression vector (P SV40 -SEAP-pA). 

Clontech pWW512

Constitutive mammalian hIFN- expression vector (P hEF1 -hIFN--pA). ( 6) pSTING Constitutive mammalian STING expression vector (P hCMV -STING-pA). IMAGE: IRAVp968F0688D pUC57 pUC19-derived prokaryotic expression vector. 

Genescript pMFO1

Custom-designed pUC57-derived vector containing the human codon-optimized PDE TBD1265 . 

This work pMFO2

Custom-designed pUC57-derived vector containing the human codon-optimized DGCA CC3285 . 

This work pMFO4

Custom-designed pUC57-derived vector containing the human codon-optimized PDE yahA . 

This work pMFO5

Custom-designed pUC57-derived vector containing the human codon-optimized DGCL. 

This work pMFO6

Custom-designed pUC57-derived vector containing the human codon-optimized mYPet-YcgR-mCyPet. This work
Marc Folcher, Sabine Oesterle, Katharina Zwicky, Thushara Thekkottil, Julie Heymoz, Muriel Hohmann, Matthias Christen, Marie Daoud El-Baba, Peter Buchmann, Martin Fussenegger
Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant