The document discusses developing engineered deep-skin bacteria that could function as beta cells in type 1 diabetes (T1D) patients by producing insulin in response to glucose levels without the need for insulin injections, using a proof of concept approach to engineer bacteria that produces a potent insulin analog, responds to glucose, and only resides in a designated individual as validated in mouse models. The engineered bacteria would utilize single-chain insulin analogs, a glucose sensor genetic circuit, and biocontainment through thymidine dependence to function like beta cells and reduce blood glucose levels in mice.

1. Needles Be Gone for T1D
Yo Suzuki, Assistant Professor
J. Craig Venter Institute

2. Long-term goal:
Make deep-skin bacterial cells that function as beta (insulin producing) cells in
T1D patients
No struggle of injected insulin therapy
No autoimmune issue of pancreas transplants
This project:
Proof of concept: a skin bacterium that (1) produces a potent insulin analog (engineered insulin),
(2) responds to glucose, and (3) resides only in a designated individual to be validated in mouse systems
Beta cells
Glucose
Insulin
Glucose
uptake
Not diabetic
Beta cells
Glucose
Insulin
Glucose
uptake
Diabetic
Beta cells
Glucose
Insulin
Glucose
uptake
Insulin
analog
Engineered
bacteria
Not diabetic

3. Bacteria live at least 3 mm into the skin in our body
in a layer of the skin previously thought to be sterile (nothing could live there)!
Platform: deep-skin bacteria
Nakatsuji et al., Nat Commun. 2013; 4: 1431.
Detection of bacteria in dermis
using Gram staining (arrows)
Advantages:
Deep-skin bacteria are in close association with capillary vessels of the skin
They are non-toxic (in a native form)
They can enter the skin in a non-invasive manner
They can be engineered to be better
They are easier to understand than human stem cells
Abundance of bacteria
Depth(mm)
Detection of bacteria in deep skin
samples using PCR (for 16S DNA)
Negative control

4. Platform: deep skin bacteria
Tool 1: potent single-chain insulin analog (engineered insulin)
Bacteria cannot make native insulin made of two peptide chains,
but it can make single-chain insulin analogs (SCIs)
SCIs that are as robust as native insulin have been developed
SCIs are not proinsulin (an intermediate in insulin production) and unlikely to share the safety concern
of proinsulin
Tool 2: glucose sensor
Genetic circuits can be constructed to make SCI production dependent on glucose
sensing
We will implement the Trz1 system, which can convert a glucose signal into the
activation of any gene via the action of three proteins
Tool 3: biocontainment
Engineered bacterial cells stay only in designated hosts
We will delete the thymidylate synthase gene and make the cells dependent on
thymidine supplied from outside
Engineered
bacteria
Thymidine application
Mouse

5. Platform: deep skin bacteria
Tool 1: potent single-chain insulin analog
Tool 2: glucose sensor
Tool 3: biocontainment
Synthetic biology brings these tools together
Constructed bacteria will be tested for their abilities to (1) induce glucose uptake
in cultured adipocytes and (2) reduce blood glucose levels in treated mice
(1) Expression of SCI
(2) Expression of 3 genes to make a glucose
sensor
(3) Deletion of the thymidylate synthase
gene
Deep-skin bacteria
Beta cells
Glucose
Insulin
Glucose
uptake
Insulin
analog
Engineered
bacteria
Not diabetic