Lecture 18 - T1D prevention and cure

Question 1

What are the clinical goals 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

Question 2

What are the disadvantages of current forms of insulin treatment?

Answer 2

Exogenous insulin is a treatment, not a cure for T1D
* Not possible to mimic physiological glucose control with insulin injections
o People with type 1 diabetes would have an HbA1c level below 7%
o Exogenous insulin therapy is not the same as a functioning pancreas (does not compensate for)
* A major problem is that blood glucose measurement and insulin administration is peripheral
* Burdensome:
o Frequent finger pricks (multiple blood glucose checks), even just to calibrate the devices and insulin injections
o Continuous glucose monitoring and insulin pumps, constant dose adjustments, psycho-social affects, meal type, physical activity
o Decreased life expectancy:
 Increased risk for hypo- and hyperglycaemic events and long-term complications
 Increased risk of death even with good glucose control

Question 3

What are mechanisms to possibly cure T1D?

Answer 3

• Halt the immune process
  o Target specific antigens
  o Target immune cells
• Replace the diseased cells/organ
  o Closed-loop system
  o Transplantation (allograft or xenograft)
  o Stem-cell derived beta cells

Question 4

What is a closed loop system?

Answer 4

• Artificial pancreas + continuous glucose sensor + insulin/glucagon dual pump
• Goal is to maintain glucose within a tight range without human interference – glucose sensor and insulin pump controlled by AI potentially or an algorithm

Question 5

What are the limitations to a closed loop system?

Answer 5

• Still unable to mimic the beta cell’s fine-tuned control of blood glucose levels
  o Room to improve pump/algorithm, insulin, sensor
• Practical issues
  o Cost and insurance coverage
  o Skin sites for sensor and pump (contact dermatitis, irritation, hair/sweat)
  o Insulin delivery (leakage, tube kinking or blockage) – constant maintenance on the system
  o Still needs human interaction (technology averse, alarm fatigue)
  o Design (personal preference against tube attachment)

Question 6

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

Answer 6

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 7

What are some benefits and limitations of intensive insulin treatment?

Answer 7

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

Question 8

What is an open loop system? What is its goal?

Answer 8

• Background insulin is pre-set
• Maintain glucose within a tight range by frequent adjustments of insulin administration based on continuous glucose monitor
• Meals still need to be “announced” – telling the insulin pump how much the dose should be based on the carbohydrate dose
  Ultimate goal: develop a beta-cell replacement therapy that will safely restore normal glycaemic control fully independent of exogenous insulin

Question 9

How to improve adoption of artificial pancreas?

Answer 9

> 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 10

What are beta cell replacement requirements?

Answer 10

• A cell source of highly functioning insulin-producing cells – preferably unlimited
• A strategy to protect the implanted cells from both alloimmune and autoimmune mediated destruction
• An optimal implantation site

Question 11

What is cell therapy? (The natural way to glucose normalisation)

Answer 11

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 12

Indications for Islet Allotransplantation

Answer 12

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 13

Pancreatic islet isolation and transplantation procedure

Answer 13

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 14

Steps for Islet Isolation

Answer 14

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 15

What are the issues of transplantation?

Answer 15

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 (to prevent clotting)
Induction during immunosuppression

Transplant rejection and autoimmune destruction

Question 16

What are the pros of islet allotransplantation?

Answer 16

• Approx. 50% of recipients have achieved independence from exogenous insulin at one year after transplants
• Glycaemic control is improved even when insulin independence is not achieved
• Effectively treats hypoglycaemia unawareness – freedom from severe hypo events

Question 17

What are the cons of islet allotransplantation?

Answer 17

• Immunosuppression required to prevent allo- and auto-immune rejection of transplanted islets
• Longevity of islet graft function (eventual loss of function)
• Expensive
• Limited availability of organ donors: quantity and subsequent quality of isolated islets

Question 18

Classification of beta-cell graft function

Answer 18

Normoglycemia: This refers to a state in which blood glucose levels are within the normal range without the need for exogenous insulin therapy. Normoglycemia is the optimal outcome of beta-cell transplantation, indicating successful graft function.

Partial function: This refers to a state in which the transplanted beta cells are producing some insulin, but not enough to maintain normoglycemia without the need for exogenous insulin therapy. Partial function can result from inadequate numbers of beta cells transplanted, immunological rejection of the graft, or other factors that impair beta-cell function.

Insulin independence with low-dose immunosuppression: This refers to a state in which the recipient is able to maintain normoglycemia without the need for exogenous insulin therapy, but requires low-dose immunosuppressive drugs to prevent rejection of the graft.

Insulin independence without immunosuppression: This refers to a state in which the recipient is able to maintain normoglycemia without the need for exogenous insulin therapy or immunosuppressive drugs. This is the most desirable outcome of beta-cell transplantation, as it indicates successful engraftment and long-term function of the transplanted beta cells.

Classification of beta-cell graft function is typically based on the recipient’s need for exogenous insulin therapy and the dose of immunosuppressive drugs required to prevent rejection of the graft.

Question 19

Animal organs: Why are pigs used as a source of islets?

Answer 19

• Organ size, physiology and function is similar to humans
• High reproductive capacity
• High donor consistency (gestation < 4 months, large litters)
• High donor consistency (inbred lines available)
• Low risk of transfer of infections (defined pathogen-free)

Question 20

How can we prevent the rejection of pig organs?

Answer 20

However, pig organs and tissue trigger a powerful rejection response in humans – xenorejection response
* Research is turning towards genetically modified (transgenic, gene knockout) pig islets to prevent xenorejection

Question 21

How are plurpotent stem cells a source of beta cells?

Answer 21

Directed differentiation

There are two main types of pluripotent stem cells that can be used as a source of beta cells: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs are derived from embryos and have the potential to differentiate into any cell type in the body. iPSCs, on the other hand, are generated from adult cells that have been reprogrammed to a pluripotent state using genetic manipulation (via CRISPR/Cas 9 gene editing)

To generate beta cells from pluripotent stem cells, researchers typically follow a multi-step process. First, the stem cells are exposed to growth factors and signaling molecules that promote their differentiation into endodermal cells, which give rise to the pancreas. Next, the endodermal cells are exposed to additional growth factors and signaling molecules that promote their differentiation into pancreatic progenitor cells, which have the potential to differentiate into all the different cell types found in the pancreas, including beta cells. Finally, the pancreatic progenitor cells are exposed to additional growth factors and signaling molecules that promote their differentiation specifically into beta cells.

Question 22

What are the limitations and safety of stem cell-derived insulin producing cells?

Answer 22

• Difficult to develop methods that fully differentiate human stem cells into mature beta cells
  o Needs to be scalable (in large quantities) and reproducible at multiple sites
  o Approved by government regulators
• Difficult to eliminate progenitor cells contaminating the final cell preparation
• The microenvironment of the transplant site may affect differentiation of progenitor cells
  o Excessive cell growth and possible tumour formation
  o Generation of off-target cell types (not beta cells, e.g., acinar cells)

Question 23

What is encapsulation: current immunotherapy to prevent beta cell destruction

Answer 23

The approach involves encapsulating beta cells in a protective coating that prevents them from being attacked by the immune system, while still allowing them to release insulin into the bloodstream to regulate blood glucose levels.

Micro-encapsulation and macro-encapsulation

Both can be removed if something goes wrong

Question 24

What are the requirements for encapsulation success?

Answer 24

• Biocompatible
• Provide ample blood supply
• Provide immune protective environment
• Allows secreted insulin to rapidly exit device and enter systemic circulation
• Retrievable if cells within are no longer functioning, tumorigenic or if other problems develop

Question 25

What are two major issues with encapsulation?

Answer 25

Biocompatibility: The encapsulation material must be biocompatible and not trigger an adverse immune response or inflammation. If the encapsulation material is not biocompatible, it could lead to the encapsulated cells being attacked by the immune system or causing inflammation that can impair their function.

Long-term viability and function of the encapsulated cells: Over time, the capsule may become thicker and limit the diffusion of oxygen and nutrients to the encapsulated cells, leading to a decline in their viability and function. Additionally, the encapsulation process may damage the cells, which could also affect their viability and function.

Question 26

What are the requirements for beta cell replacement as a cure for T1D?

Answer 26

• Need a cell source: cadaveric pancreatic islet, procine pancreatic islets or stem cells
• New drug regimens to improve graft survival
• Protect beta cells from destructive allo-, xeno-, or auto-immune responses
  o Encapsulation
  o Genetic engineering via CRISPR/Cas9 gene editing
• A graft site that is optimal for beta-cell survival and function:
  o Oxygenation
  o Nutrient exchange
• Requirement for other pancreatic cells to support differentiation and/or function of beta cells

Question 27

What are the requirements for immune-based therapy?

Answer 27

• Safety
  o Patients are primarily children
  o existing insulin therapy is relatively safe
• Effective
  o At least retain remaining beta cell mass/function
• Durable
  o Preferable not to need lifelong treatment

Question 28

How can beta cell autoimmunity be prevented via the mucosal tolerance approach?

Answer 28

The approach involves exposing the immune system to beta cell antigens via the mucosal surfaces of the body, such as the nose, mouth, and gut, in order to induce immune tolerance and prevent autoimmune attacks on beta cells.

Mucosal tolerance: the cells lining the nose and gut process foreign antigens and bring about clonal deletion or anergy of T cells and induces regulatory T cells

Question 29

How can islet autoantibody targets be potential therapeutic antigens for T1D?

Answer 29

• Components of the insulin secretory vesicle are targets of autoantibodies in individuals with T1D
• Antigens targets of T cells overlap with autoantibody targets as well as other regions of the islet
• Blocking the insulin-specific T cell responses in the NOD mouse prevents diabetes

Question 30

What are some immune targets in stage 3 of T1D/clinical diabetes that have delayed beta-cell decline?

Answer 30

CD3: CD3 is a protein that is expressed on the surface of T cells, and is involved in T cell activation. A monoclonal antibody against CD3 called OKT3 has been shown to delay the decline in beta cell function in individuals with new-onset T1D. TEPLIZUMAB

CD20: CD20 is a protein that is expressed on the surface of B cells. A monoclonal antibody against CD20 called RETUXIMAB has been shown to decrease the number of autoreactive B cells and delay the decline in beta cell function in some individuals with T1D.

CTLA-4: CTLA-4 is a protein that is expressed on the surface of T cells, and is involved in regulating T cell activation. A fusion protein that contains CTLA-4 called ABATACEPT has been shown to delay the decline in beta cell function in some individuals with T1D.

IL-1: IL-1 is a cytokine that is involved in inflammation and immune responses. A monoclonal antibody against IL-1 called CANAKINUMAB has been shown to delay the decline in beta cell function in some individuals with T1D.

Interferon-alpha: Interferon-alpha is a cytokine that is involved in immune responses. A monoclonal antibody against interferon-alpha called SIFALIMUMAB has been shown to delay the decline in beta cell function in some individuals with T1D.

Question 31

What are the challenging aspects of antigen-specific immunotherapy?

Answer 31

• Target antigen
• Route of delivery
• Timing of therapy
• Surrogate end-point/biomarker

Question 32

How can we halt immune-mediated beta cell destruction by JAK inhibitors?

Answer 32

• The JAK/STAT pathway is a critical intracellular signalling mechanism for responding to inflammatory cytokines
• AZD1480 and Baricitinib: inhibitors of JAK1 and JAK2 that block cytokine signalling pathways in immune cells and beta cells
• Blocking the effects of cytokines on immune cells and beta cells will reverse new onset type 1 diabetes

Question 33

Explain halting immune-mediated beta cell destruction: Co-stimulation blockade

Answer 33

Co-stimulation blockade is a type of immunotherapy that aims to halt the immune-mediated destruction of beta cells in type 1 diabetes (T1D). The therapy works by blocking the interaction between T cells and antigen-presenting cells (APCs), which are immune cells that present antigens to T cells and activate them.

T cell activation requires two signals: one signal is provided by the presentation of an antigen on an APC, and the second signal is provided by a co-stimulatory molecule on the APC. Co-stimulation blockade aims to block the second signal by inhibiting the interaction between the co-stimulatory molecule on the APC and the corresponding receptor on the T cell.

One co-stimulatory molecule that has been targeted in T1D is CD28, which is expressed on the surface of T cells, and its ligands, CD80 and CD86, which are expressed on the surface of APCs. A fusion protein called CTLA-4Ig, which consists of the extracellular domain of the T cell receptor CTLA-4 fused to the Fc portion of immunoglobulin G (IgG), can bind to CD80 and CD86 and block their interaction with CD28. This prevents T cell activation and the subsequent destruction of beta cells.

Question 34

What is the therapy abatacept?

Answer 34

It is a fusion protein composed of the extracellular domain of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the Fc portion of immunoglobulin G1 (IgG1).

Abatacept works by inhibiting the interaction between antigen-presenting cells (APCs) and T cells, which is a key step in the activation of the immune response. Specifically, abatacept binds to CD80 and CD86, which are co-stimulatory molecules expressed on APCs, preventing their interaction with CD28 on T cells. This results in decreased T cell activation and a reduction in the immune response.

Question 35

What is the therapy Teplizumab?

Answer 35

• Blocking or providing weak agonistic stimulation via CD3 will eliminate pathogenic T cells and/or induces regulatory responses
• The CD3 complex associates with the T-cell receptor to bind to MHC/peptide complexes on APCs and initiates the intracellular activation signal in T cells
• Teplizumab is a humanised anti-CD3 monoclonal antibody that provides weak agonistic T cell stimulation associated with anergy and regulatory responses

Question 36

How can we halt immune-mediated beta cell destruction by altering the t-cell response?

Answer 36

Immune tolerance induction:

T-cell receptor redirection:

Co-stimulation blockade:

Regulatory T cells:

Antigen-specific immunotherapy:

Question 37

How can we halt immune-mediated beta cell destruction by through regulatory T cells?

Answer 37

Treg expansion and reinfusion: Tregs can be expanded in vitro using growth factors and then reinfused into the patient. This approach has been shown to be effective in preclinical studies, but it is difficult to achieve sufficient numbers of Tregs for clinical use.

Treg induction in vivo: Tregs can also be induced in vivo using immunomodulatory agents such as interleukin-2 (IL-2) or anti-CD3 monoclonal antibodies. These agents promote the differentiation and expansion of Tregs, which can then help to suppress the immune response.

Treg-specific therapies: Treg-specific therapies are designed to specifically target and activate Tregs. For example, low-dose interleukin-2 (IL-2) therapy has been shown to selectively expand Tregs in patients with T1D, which may help to prevent beta cell destruction.

Gene therapy: Tregs can be engineered to express genes that promote their activity or target them specifically to the site of inflammation. For example, Tregs can be engineered to express IL-10, which is a cytokine that helps to suppress the immune response.