Type 1 Diabetes (6)

Question 1

Clinical goal for individuals with recent onset and long-standing T1D

Answer 1

To improve metabolic outcomes and quality of life for people living with the daily challenges and long-term risk of complications associated with T1D

> life expectancy reduced by about 10 years on avergae

Question 2

Recall lecture 4: Exogenous insulin is a treatment, not a cure for T1D

Answer 2

Optimal glycaemic control requires:

> multiple BGL measurements 4x a day
multiple-dose insulin regimen (>4x injections/day) to mimic physiological insulin release

> detecting hypoglycaemia events (lollies)

Question 3

Beta cell substitution

Answer 3

Manual to hybrid-loop systems of insulin delivery

> insulin pumps etc

> unless inventions are shown to create value by improving outcomes like improving life expectancy, the govt is unlikely to fully fund the new tech that comes along to manage diabetes unless it can be proven to be much better than conventional therapy

Question 4

Glucose levels of a child with T1D using a conventional insulin pump

Answer 4

Detection by continuous blood glucose monitoring (day and night)
>difficult to maintain normo-glycaemic levels even with automatic infusion of exogenous insulin (i.e. insulin pump)

Question 5

Intensive insulin treatment: less complications, but more hypoglycaemia

Answer 5

As HbA1c levels increase,
>rate of severe hypo decreases
>rate of progression of retinopathy increases

Question 6

Disadvantages with current treatment?

Answer 6

Even with continuous glucose monitoring and insulin pumps:
>frequent dosing adjustments (meal type, physical activity, illness, stress)
>not possible to mimic physiological glucose control

<30% of people with T1D achieve recommended glucose control
>maintain glucose levels within target range
>maintain HbA1c under target threshold (<7.5% for children, <7% in adults)
>Increased risk for hypoglycaemia events and long-term complications

Uniquely burdensome
>inject multiple times per day or maintain insulin pump
>ongoing collaboration of machines and algorithms (still have daily finger pricks)

*Exogenous insulin treatment falls well short of a cure

Question 7

Artificial Pancreas: Closed-loop system for T1D

Answer 7

Artificial pancreas controller + continuous glucose sensor + insulin/glucagon dual pump

> hard to determine potential of these new systems for 2 reasons

1) companies constantly pushing out new systems, old ones being studied, hard to understand true potentials
2) most people that are being tested on have well controlled diabetes, and not tested on real world cohorts of patients with poor control of diabetes

*big step forward in BGL management but still dont represent a cure

Question 8

Artificial Pancreas: Limitations that need to be addressed

Answer 8

One of the barriers for working out the potentials of these devices is the risk aversion of the companies that make these devices
>reluctant to push the algorithms for very tight glucose control levels in case they cause problems
>Biohacker movement where people are re-jigging the algorithms of the devices off label to try and make these devices work better and have tighter glucose control

Question 9

Adoption of an artificial pancreas: Potential and Limitations

Answer 9

Potential
>63% use insulin pumps
>30% use continuous glucose monitors
>82% of parents/caregivers reported devices made it easier for their family members to achieve health targets

Limited adoption due to:
>cost of device
>insurance obstacles
>hassle of wearing devices

Other
>skin related issues (e.g. contact dermatitis)
>Types and duration of physical activity
>discontinuity/disruption of insulin delivery
>kinking or blockage of the deliver tubes
>leakage from the site
>bruising and bleeding

Question 10

How to improve adoption of artificial pancreas?

Answer 10

> miniturising the devices and improving wearability through innovations
incorporating implantable components
incorporating inputs beyond glucose concentration
taking advantage of big data analysis and machine learning algorithms

Question 11

The ultimate goal: glucose control without external devices

Answer 11

Develop a beta cell replacement therapy that will safely restore normal glycaemic control fully independent of exogenous insulin, thereby eliminating the burden of insulin use for people with T1D

> 2 things have to be addressed:
beta cell mass
autoimmune response

Question 12

Cell therapy: The ‘natural’ way to glucose normalisaiton

Answer 12

Cadeveric Donor Islets > islet isolation > transplant direcetly into patient > immunosuppression, tolerance

*once stage 3 T1D, not enough beta cells to produce enough insulin, at Stage 1 and 2 still can try and preserve the remaining beta cells

Requirements:

1) Cell source of highly functioning insulin-producing cells
2) Strategy to protect the implanted cells from alloimmune and autoimmune-mediated destruction
3) an optimal implantation site

Question 13

Indications for Islet Allotransplantation

Answer 13

Who is eligible to receive a transplant?
(islet transplant makes more sense than whole pancreas transplant, only 2% of your cells non-functional)
>Adults with T1D having problematic hypuglycaemia unawareness
>Adults, T1D, having kidney transplant if unsuitable for whole pancreas (usually whole pancreas along with kidney transplant)
>Adults, T1D, hypoglycaemia unawareness but responsive to conventional treatment
>Individuals with other types of beta cell failure: MODY, T2D

How many islets are needed?
>typically >1 transplant (infusion) is required per recipient to become insulin independent
>10000-12000 islet equivalents per kg of body weight

Question 14

Pancreatic islet isolation and transplantation procedure

Answer 14

1. Donor pancreas
2. Ricordi Chamber: key islet isolation device
3. Separated islets
4. Islets are introduced into the recipient liver
5. Transplanted islets secreting insulin in the liver
   (put into portal vein that drains gut into liver, via radiology procedure, cannot put in pancreas)

Question 15

Steps for Islet Isolation

Answer 15

1) Pancreas harest
>retrieval
>organ preservation

2) Organ preparation
>cleaning
>duct annulation
>enzyme injection

3) Isolation
>enzymatic digestion
>mechanical digestion

4) Purification
>filtration
>density separation

5) Culture
>plating quality control
-viability
-count
-sterility

Question 16

Issues of transplantation

Answer 16

One big issue is the need for immunosuppression in any form of allo-transplantation (from anyone who is not an identical twin)

• need to transplant into liver - use heparin as anticoagulant
• induction during immunosuppression

Question 17

Islet transplant patient results

Answer 17

Evolution vs Technology - in an islet transplant pt

With transplantations, HbA1c and BGL is highly improved, this particular PT could eventually stop taking exogenous insulin

Not putting in something that is new, putting in something from someone who is dead, so there is issues around the efficacy of the implanted tissue

Question 18

Classification of beta-cell graft function

Answer 18

Functional status:
>failure if C-peptide level is baseline

Optimal, good, and marginal status all have c-peptide levels above baseline

Question 19

Evaluation of Patients Receiving Islet Transplant

Answer 19

Showed that hypoglycaemia is very successfully controlled in this way, even when insulin is required

Question 20

Artificial Pancreas vs Islet Transplant

Answer 20

Glucose control better in islet transplant

Question 21

Pancreatic islet allotransplantation: Pros and Cons

Answer 21

Pros
>islets can be isolated at multiple sites using a common manufacturing process
>50% of recipients achieve independence from exogenous insulin at one year from transplant
>Glycaemic control is improved even when insulin independence is not achieved
>Effectively treats hypoglycaemia unawareness - freedom from severe ‘hypo’ events

Cons
>limited availability of organ donors: quantity and subsequent quality of isolated islets
>immunosuppression required to prevent allo- and auto-immune rejection of transplanted islets
>longevity of islet graft function (eventual loss of function)
>expensive (>$140,000 per isolation/transplant) - procedure not covered by health insurance

Question 22

Future sources of islets and beta cells for transplantation

Answer 22

Caderveric Donor Islets

Porcine Islets (genetic modification)

Pluripotent Stem Cells
(differentiation, manufacturing)

What we really need is an unlimited supply of insulin producing cells, potential sources are stem cells or animal islets

Other element that is potentially possible in the future is regeneration of the insulin producing cells that remain- hard especially in adults

Question 23

Animal organs: Why the pig?

Answer 23

Organ size, physiology and function similar to humans

High reproductive capacity (gestation <4 months, large litters)

High donor consistency (inbred lines available)

Low risk of transfer of infections (defined pathogen-free)

Pig organs and tissues trigger a powerful rejection response in humans (xenograph immune response is extremely strong but there has been progress)

Question 24

Preventing Rejection of Pig Organs

Answer 24

Immunosuppress the patient
>drug side effects
>risk of infection
>risk of cancer
>cost benefit has to be there for the pt for immunosuppressants
>also calcineurin inhibitors suppress kidney function

Genetically modify the donor pig
>make them immunologically inert so no immunosuppression is required
>CRISPR/Cas9 genome editing
>delete pig genes encoding ‘xenoantigens’
>Add human genes to regulate the human immune response
>remove porcine endogenous retroviruses

Question 25

Transgenic and knockout pigs are being tested for xenotransplantation

Answer 25

Transgenic and knockout pig islets currently being tested in diabetic baboons by groups at st vincent’s hospital and westmead institute

Question 26

Other sources of beta cells: Human pluripotent stem cells?

Answer 26

Generate functional human beta cells in vitro
>CRISPR/Cas9 genome editing of hPSC (Oct4+)
>definitive endoderm (Sox17+)
>Pancreatic progenitor (Pdx1+)
>Endocrine progenitor (Ngn3+)
>Beta-like cell

> stem cells are not fully mature beta cells, requires in vivo maturation
big difference in the cell iwhich produces some insulin vs a proper beta cell which is a factory of insulin
(we are not yet able to reproduce the process to make a full beta cell, can only make cells that produce SOME insulin)
were able to secrete insulin in response to glucagon stimulation

Question 27

Stem cell-derived insulin producing cells: Limitations and safety

Answer 27

Difficult to develop methods that fully differentiate human stem cells into mature beta cells

• needs to be scalable (i.e. generate large quantities)
• reproducible at multiple sites
• approved by government regulators

Difficult to eliminate progenitor cells contaminating the final cell preparation

The micro-environment of the transplant site may affect differentiation of progenitor cells

• excessive cell growth and possible tumor formation
• generation of off-target cell types (i.e. not beta cells, e.g. acinar cells)

Question 28

Encapsulation

Answer 28

One way to avoid immunosuppression when doing cell transplants is to use a physical barrier to the immune system by the form of a bioengineered capsule around the transplant
-microcapsule where all the transplanted cells are placed into
>encapsulation devices can be removed if somehthing goes wrong

Question 29

Encapsulation requirements

Answer 29

Biocompatible

Provide ample blood supply

Provide immune protective environment

Allow secreted insulin to rapidly exit device and enter systemic circulation

Contain any potentially tumorigenic cells

Retrievable

Question 30

2 major issues with capsulation

Answer 30

1) failing of the biomaterial
>foreign material in body will always cause immune response

2) Physiologically, big issue of o2 delivery
>barrier increases diffusion distance and lowers oxygen tension around the cell

Question 31

Summary: Major considerations for beta cell replacement as a cure for T1D

Answer 31

Cell source: cadaver pancreatic islets, porcine pancreatic islets, or stem cells

New drug regimens to improve graft survival

Protect beta cells from destructive allo-, xeno- or auto-immune reponses
>encapsulation
>genetic engineering via CRISPR/Cas9 gene editing

A graft site that is optimal for beta-cell survival and function
>oxygenation
>nutrient exchange

Requirement for other pancreatic cells (e.g. mesenchymal cells) to support differentiation and/or function of beta cells

Question 32

Still need to stop immune cells from destroying beta cells

Answer 32

Prevention
>prevent beta cell autoimmunity
(antigen-specific tolerance)

> halt beta cell attack and save remaining beta cells
(immune therapeutics)

Treatment
>restore beta cell function
(islet and pancreas transplants)

> improve glucose control
(artificial pancreas)

Question 33

Requirements for Immune-based therapy

Answer 33

Safety
>patients are primarily children
>existing insulin therapy is relatively safe

Effective
>must be at least retention of remaining beta cell mass/function

Durable
>preferable not to need lifelong treatment

Question 34

Prevent beta cell autoimmunity: Mucosal tolerance approach

Answer 34

Underlying concept:
>mucosal tolerance: the unique lining of the gut processes foreign antigens and brings about clonal deletion or anergy of T cells and induction of Tregs
> how can we use this concept to get tolerance for our own cells?

> clinical trials using insulin as the autoantigen failed to reverse diabetes
other clinical trials used GAD, but also failed to decrease or delay T1D incidence

Question 35

Halting immune-mediated beta cell destruction: B-cell depletion

Answer 35

Underlying concept:
>B cells produce autoantibodies, act as APCs to activate autoreactive CD8+ T cells
>depleting B cells may delay disease iin at-risk individuals or reverse disease in new onset cases
>CD20, a cell surface protein on B cells required for activation and proliferation (not expressed on long-lived plasma cells)
>Rituximab - CD20-specific monoclonal antibody that depletes B cells in vivo via antibody-dependent cellular cytotoxicicity (ADCC) and complement-dependent cytotoxicicity (CDC)

Question 36

Clinical trials with Rituximab (CD20 monoclonal Ab)

Answer 36

Reduced insulin-specific & GAD65-specific autoantibodies

> T cell proliferative response to islet antigens were not reduced

1 year post treatment
>delay in insulin loss (measured by C-peptide), lower HbA1c, and reduced exogenous insulin usage

2 year post treatment
>no reduction in HbA1c or exogenous insulin usage compared to placebo group

Question 37

Halting immune-mediated beta cell destruction: Co-stimulation blockade

Answer 37

Underlying concept:
>Activation of naive T cells requires TCR interaction with peptide/MHC presentation by APCs AND co-stimulatory signals
>CD28-CD80/86 co-stimulation activates T cell responses
>CTLA4-CD80/86 co-stimulation inhibits T cell responses

> Abatacept - chimeric protein composed of human CTLA4 fused to a modified human IgG1 Fc acts as decoy receptor for CD80/86 and blocks Cd28-CD80/86 induced co-stimulation

Question 38

Therapy: Abatacept

Answer 38

Preserved c-peptide levels for up to 9.6 months, indicating delayed disease progression

2 year post treatment
>higher c-peptide, lower HbA1c, reduced exogenous insulin usage compared to placebo group

3 year post treatment
>lower HbA1c
>similar exogenous insulin usage to placebo group

Question 39

Halting immune-mediated beta cell destruction: alter the t-cell response

Answer 39

Underlying concept:
>blocking or providing weak agonistic stimulation via CD3 will eliminate pathogenic T cells and/or induce regulatory responses
>the CD3 complex associates with the T-cell receptor (TCR) to bind to MHC/peptide complexes on APCs and initiates the intracellular activation signal in T cells

> Teplizumab - humanised anti-CD3 monoclonal antibody that provides weak agonistic T-cell stimulation associated with anergy and regulatory responses

Question 40

Therapy: Teplizumab

Answer 40

Possible to preserve c-peptide levels for 2-4 years indicating delayed disease progression

Possible to identify responders: younger patients treated within 6 week diagnosis

Associated with increased PD-1+ exhausted and anergic CD8+ T cells

Did not reduce HbA1c or exogenous insulin usage

Question 41

Halting immune-mediated beta cell destruction: regulatory T cells

Answer 41

Underlying concept:
>increasing the number of regulatory T cells (Foxp3+) will increase T-cell tolerance to islet antigens and reduce beta cell destruction
>Tregs - specialised T-cell subpopulation that express high levels of FOXP3 and CD25. Suppress immune responses via:
>expression of CD25: prevents proliferation/differentiation of effector T cells
>Secretion of immunosuppressive factors: IL-10, IL-35, TGFB
>Cell-to-cell contact: CTLA4 expression and granzymes

Question 42

Therapy: Autologous Treg Cell Transfer

Answer 42

Expansion of Tregs from peripheral blood resulted in Tregs with increased suppressive activity in vitro

After infusion in subjects:
>no adverse events
>25% of infused cells still present by 9 months
>no decline in endogenous insulin production - c-peptide levels unchanged out to 1+ years

Phase II trial underway