Presentation 6: Green Tea Genetic Control

Question 1

What is inducible gene switching?

Answer 1

Rapid activation of gene transcription to produce new proteins in response to external stimuli
• Applied in cell therapy
• Translation into clinical use is limited

Question 2

What can trigger inducible gene switching?

Answer 2

Antibiotics
Food or cosmetic preservatives
Caffeine

Question 3

What is an ideal trigger molecule for inducible gene switching?

Answer 3

Natural
Nontoxic
Highly soluble
Inexpensive
Perhaps beneficial

Question 4

What is PCA?

Answer 4

Green tea metabolite
Antioxidant
Engineered beverage-triggered remote control transgenic orthogonal switches triggered by PCA
PCA is a remote-control inducer that is safe and tightly regulated.

Question 5

What is the PCA on switch?

Answer 5

Synthetic transrepressor (PcaR) created by fusion of the KRAB domain to N terminus of PcaV.
PcaR binds and silences gene expression from synthetic promoter PPcaR7 linked to a tandem OPcaV binding site.
In the presence of PCA, repression is disrupted, and the constitutive gene is expressed, in its absence, the gene remains repressed by PcaV.
• Production of SEAP in the presence of PCA acts as a validation for the PCAON gene switch

Question 6

How is the PCA off switch created?

Answer 6

PCAOFF turns “OFF” transgene expression (SEAP) in the presence of PCA
Composed of Psv40, PcaA (PcaV + VP16), pA, Ppcaa5 ((Opcav)5) + Phcmvmin), SEAP
PCA nullifies the repression of (Opcav)5 (repressor) by PcaA
Used in VA NIMPLY PCA, NOR gates

Question 7

How is the PCA ON gene switch optimized?

Answer 7

Vital to choose the right combination of synthetic PcaR-specific promoters and constitutive promoters for driving gene expression.
The goal was to identify a combination that yielded maximal gene repression in the absence of PCA and maximal gene induction in the presence of PCA.
Tested many different combination variants in vitro in mammalian cells used to find the right combination: PcaR+PPcaR7 (G)showing the highest expression performance.

Question 8

How was switch validation tested?

Answer 8

Ligand Specificity of PcaRON
Testing of ligand specificity ensures that chemical analogues of PCA were unable to cross-activate the designed PCAON system
Analogous compounds were found to have no significant cross-activation of the PCAON switch.

Question 9

How was dose and time dependence for the PCA switch tested?

Answer 9

Showing increased fold induction of SEAP with increased concentration of PCA intake.

Question 10

How was the reversibility of the PCA on switch tested?

Answer 10

Reversibility of of HEKPCA-ON-SEAP-mediated SEAP expression was exhibited by alternating daily dosage with periodic concentration testing.
Able to verify and ON-OFF-ON reversibility as well as an OFF-ON-OFF reversibility system.
The good performance of HEKPCA-ON-SEAP as a fully reversible and dosage tunable SEAP output over this trial verified the transgene’s ability as a suitable gene switc

Question 11

How was PCA-induced and gRNA-mediated control of CRISPR-Cas9 activity determined?

Answer 11

Engineered 3 PCA inducible CRISPR systems

1) Gene inhibition
2) Gene activation
3) Gene deletion

Question 12

How was PCA-induced and gRNA-mediated control of CRISPR-Cas9 activity designed?

Answer 12

𝑂𝑃𝑐𝑎𝑉 binding sites upstream and down stream of U6
Engineered PcaR protein bind to U6 adjacent sites
• PCA disrupts PcaR binding and activates U6-driven gRNA expression
• SEAL reporter used for optimization

Question 13

How was PCA-induced and gRNA-mediated control of CRISPR-Cas9 activity designed for inhibition?

Answer 13

Inhibition device mechanism
CAG promotor express PcaR
PcaR bind to U6 adjacent sites; gRNA expression repressed
PCA induces expression of gRNA
gRNA combines with constitutively expressed
dCas9-KRAB
gRNA-dCas9-KRAB repressive complex
Binds to specific DNA sites to inhibit gene expression

Question 14

How was PCA-induced and gRNA-mediated control of CRISPR-Cas9 activity designed for activation ?

Answer 14

Activation device mechanism
Promotor containing 5 OpcaV repeats both up and down stream of U6 promoter
PCA-inducible scaffold gRNA expression for the synergistic activation of mediator complex (SAM)
gRNA and SAM recruits Cas9 to form transactivator complex
Enable target gene activation

Question 15

Explain the gRNA dependent gene editing

Answer 15

For Gene deletion
To test endogenous gene deletion
Frameshift enhanced green fluorescent protein (fsEGFP)
GFP expressed when coding frame is restored by error prone nonhomologous end joining

Question 16

What do biocomputers rely on?

Answer 16

Biocomputers rely on biomolecule inputs, inducing or repressing transgene expression (output)

Question 17

How were the biocomputers used?

Answer 17

Effect of the biomolecule inputs on transgene expression is dependent on the algorithm (configuration of gene switches, promoters, repressors, transactivators and transrepressors)
Input signal is PCA and VA (vanillic acid), output signal is SEAP (in mice) or d2EYF (in mammalian cells) expression
Two inputs (PCA and VA) were required for NIMPLY, AND, OR, NOR logic gates

Question 18

How does the PCA NIMPLY VA Logic GATE work?

Answer 18

Transgene expression occurs if only PCA is present
Composed of PCAON switch, VAOFF switch and a hybrid promoter PPV1
If PCA is present, PcaR repression of (Opcav)2 is nullified
If VA is present VanA5 activation of P1VanO2 is lost
Transgene expression requires P1VanO2 and (Opcav)2 to be active

Question 19

How does the VA NIMPLY PCA Logic GATE work?

Answer 19

Transgene expression occurs if only VA is present
Composed of PCAOFF, VAONgene switches and hybrid promoter PPV2
If PCA ispresent Pca Aactivation ofPPcaA1i is lost
If VA is present VanA4 repression of VanO4 is
nullified
Transgene expression requires bothPPcaA1 and VanO4 to be active

Question 20

How does the AND LOGIC GATE WORK?

Answer 20

Transgene expression occurs only if both PCA and VA are present
If PCA present PcaR repression of OPcaV is nullified
If VA is present VanA4 repression of VanO4 is nullified
Transgene expression requires both OPcaV and VanO4 to not be repressed

Question 21

How does the OR LOGIC GATE WORK?

Answer 21

Transgene expression occurs ifPCA or VA is present
If PCA or VA are present GV repression by PcaR or VanA4 is nullified
GV activation triggers transgene expression

Question 22

How does the NOR LOGIC GATE work?

Answer 22

Transgene expression occurs only if PCA nor VA are present
DocS targets and complexes with ligand Coh2
TetR + Docs in complex with Coh2 + VP16 triggers transgene expression
If either PCA orVA is present a co- transactivator is repressed

Question 23

How can biocomputers be applied?

Answer 23

Multiple logic gates can be used in parallel to precisely and remotely control therapeutic bioimplants

Question 24

What is the PCA ON switch 2.0?

Answer 24

Uses PcaK a PCA transporter protein, which pumps PCA into the engineered cells
Increased intracellular concentrations of PCA
Highly sensitive

Question 25

What was the PCA ON switch 2.0 able to determine?

Answer 25

A PCA ON switch version 2.0 was created which was more sensitive than version 1.0.
Compared to injection of PCA, orally administered PCA is better with respect to patience compliance.
SEAP is expressed at low PCA concentration.
PCA could control transgene expression in a dose- dependent manner, regardless of the delivery method

Question 26

What was determined when the PCA-regulated engineered cells for diabetes therapy in mice - PCA ON switch 2.0

Answer 26

No injection of PCA
Less amount of PCA is required to trigger the induction system and allow a oral treatment based on oral administration of PCA or green tea.
The lowest oral PCA dose led to sufficient improvements in insulin expression to control blood glucose homeostasis