May 2004

Report of the NIDDK/NCRR Beta Cell Working Group:

Translation Toward a Cell-based Therapy for Diabetes

**[TOP PRIORITIES]{.underline}**

[Reagents]{.underline}

-   50 usable antibodies for beta cell researchers

-   50 usable mouse strains for beta cell researchers

**[Assays]{.underline}**

-   Assays to screen chemical libraries for modifiers of beta cell
    function, regeneration, immunogenicity, differentiation, and
    expansion, with the goal of screening 100,000 compounds in two years

-   Assays to monitor allo- and autoimmune responses

-   Assays to measure beta cell mass and function *in vivo*

-   Assays to monitor potency (transplant potential) in isolated human
    islets

-   Assays for immunogenicity of islets and cell-based islet replacement
    products

> **[Products]{.underline}**

-   Microencapsulation or other functional immunobarrier

-   Devices (e.g. isolation chamber) to allow 'training' and/or testing
    of engineered neoplastic lines in the setting of a whole animal.
    These need not provide a perfect immunobarrier in order to be
    useful. They could also be used in studies of the regenerative
    potential of islets, as well as for engineering cells by training
    them to survive in the presence of a host immune system.

**[Therapies]{.underline}**

-   New agents capable of inducing local immunosuppression at the
    transplant site

-   New methods of 'cloaking' from the host immune system transplanted
    islets, engineered insulin-producing cells, and replacement cells
    derived from stem cells

-   New pathway-specific immunosuppression strategies

**[Research Climate]{.underline}**

-   There must be an avenue for developing reagents for human use that
    are not solely controlled by marketplace potential. Public or
    academic development of therapeutics might require purchase or
    rental of the intellectual property from a company. New promising
    agents can be further developed through the NIDDK-sponsored T1-RAID
    (see appendix 1).

**RECOMMENDATIONS AND STRATEGIC PLAN:**

**[Introduction]{.underline}**

NIDDK recognizes the need for developing cell-based therapies, such as
islet transplantation or engineered insulin-secreting cell products, to
treat diabetes. At this point in time (May, 2004) the diabetes research
community has at its disposal a growing body of knowledge gleaned from
basic and clinical studies; a series of funded individual research
projects in areas such as beta cell development, tolerance and
immunology, beta cell/islet physiology, and islet imaging; and a series
of resources and consortia that have been set in place over the last
five years. Many of these resources have been provided using specially
mandated funds earmarked for type 1 diabetes, supplemented with regular
research funding from The National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK), The National Institute of Allergy and
Infectious Diseases (NIAID) and The Juvenile Diabetes Research
Foundation International (JDRFI). A small advisory committee (appendix
3) was convened at NIH in February of 2004 to explore the current state
of the art in cell based therapies for diabetes, and to identify
opportunities for scientific advancement in the near future. Advice was
also gleaned from a NIH/JDRFI sponsored workshop, "Immunobarriers for
Pancreatic Islet Transplantation", March 29-30, 2004 in Washington, DC.
The intent of this report is to communicate this advice, to indicate
areas of current opportunity that might contribute to the search for a
cell based therapy for diabetes, and to suggest specific needs that
might benefit from NIH efforts.

**[I. Reagents for Beta Cell Research]{.underline}**

Basic beta cell research requires reagents and a reliable source of high
quality human islet tissue. Especially needed are antibodies to cell
surface antigens of interest, as identified by the NIDDK-funded Beta
Cell Biology Consortium (BCBC) and other scientists, that will be
suitable for isolating and characterizing beta cells and their
progenitors. Genetically marked mice are also needed for lineage
tracing, imaging, etc. Finally, although research grade human islet
tissue is now available for purchase through the Islet Cell Resources
(ICRs), issues of standardization, quality control and assurance remain
to be thoroughly addressed.

[Needed:]{.underline}

-   Widely available well-characterized antibodies to human and mouse
    islet cell antigens;

-   Genetically marked mouse models for beta cell therapy research;

-   Genetically manipulated mouse models for beta cell therapy research;

-   Reliably high quality islets available for research from the ICRs;

-   Widely accepted standards and assays for assessing islet quality.

**[II. Beta Cell Regeneration]{.underline}**

About 50% of type 1 diabetic patients eligible for islet transplant
exhibit low levels of C-peptide. There is anecdotal evidence suggesting
that endogenous insulin secretion may be restored in a subpopulation of
patients bearing failed islet transplants. Transplantation may create an
environment that nurtures beta cell regeneration by combining
suppression of the autoimmune response and a normalized glucose (and
lipid) profile. An opportunity exists to study and characterize this
regenerative potential in such patients. In addition, it appears that
recovery of beta cell mass following partial pancreatectomy occurs
through an increase in the number of beta cells in each islet, rather
than through the production of new islets. Basic research is needed to
determine the factors important for beta cell regeneration and to
improve this ability. Appropriate animal models are needed.

[Needed:]{.underline}

> **II.1. Patient Studies**

-   Islet transplantation patients should be monitored for endogenous
    insulin secretion;

    -   Patients in the islet clinical trials could be monitored for
        evidence of regeneration using a common set of criteria for
        identifying them;

    -   Patients that are identified with potential beta cell
        regeneration might be further studied at a common site;

-   Type 1 diabetic patients undergoing immune suppression without islet
    transplantation (kidney or liver transplant recipients) should also
    be monitored for beta cell regeneration;

-   Patients undergoing partial pancreatectomy for non-diabetic diseases
    could be monitored for evidence of beta cell and islet regeneration;

-   Regeneration might be studied in small pilot clinical trials by
    treatment with targeted immunosuppression of long term type 1
    diabetic patients (perhaps candidates for islet transplantation,
    prior to therapy) who test positive for C-peptide. Increased
    C-peptide could easily be observed in these patients. This might be
    too risky for new onset patients, especially children;

-   Studies should be designed to evaluate C-peptide levels over time in
    new onset type 1 diabetes, perhaps starting in children identified
    as at high risk for type 1 diabetes because of HLA class and the
    presence of autoantibodies;

-   Determine the incidence of C-peptide expression in type 1 diabetics;

-   Imaging must be developed as a tool to monitor regeneration in
    patients.

> **II.2. Animal Studies**

-   Animal models, such as non-human primates transplanted with human
    islets can be used to identify exogenous or endogenous factors
    (i.e.,GLP-1) that might promote beta cell regeneration;

-   Partial pancreatectomy followed by transplantation of isolated
    islets into the liver of the same animal (dog, non-human primate) or
    a new host might prove a good model for the study of regeneration of
    in situ and transplanted islets in a non-autoimmune environment;

-   Models where a rapid change in body weight produces compensatory
    changes in beta cell mass (pregnancy, fat feeding) should be studied
    to understand the factors that control regeneration and expansion of
    islets and beta cells;

-   Models of type 1 diabetes that more closely resemble human disease
    are needed.

> **II.3. Basic Research**

-   Basic research is needed that leads to a more complete understanding
    of beta cell life cycle and regeneration;

-   Chemical libraries should be screened for compounds that induce beta
    cell replication and regeneration.

**[III. Tolerance and Immunosuppression]{.underline}**

Development of new drugs or strategies to promote immunosuppression is a
primary roadblock to achieving effective tolerance, graft survival, and
islet regeneration. Intellectual property (IP) and patent issues may
have a profound and often negative impact on the development and testing
of promising drugs by companies, and may impede access to compounds by
academic researchers. Solutions can sometimes be found, such as short
term purchase of IP, but this can be very expensive. The Type 1-RAID
program, sponsored by NIDDK and NCI, may help investigators develop
promising new drugs and biologics (see appendix 1).

Interventions are needed to block specific immune pathways. Methods for
local immunosuppression at the site of transplantation are needed and
should be explored. New animal models, such as human islets transplanted
into mice or nonhuman primates, are needed to study immunosuppressive
therapies.

[Needed:]{.underline}

-   Tolerizing agents that target specific pathways, such as the
    CD40:CD40L pathway;

-   Methods for inducing localized immunosuppression;

-   New ideas, animal models, methods and clinical trial designs for
    testing immunotherapies[;]{.underline}

-   Studies to determine why anti-CD154 treatment leads to
    thromboembolic problems and how to avoid this side effect.

**[IV. Islet Transplantation]{.underline}**

An important goal for islet transplantation should be to achieve a 1:1
donor to recipient ratio. This might be achieved by improvement of
isolation and quality testing of islets, or by using live partial
pancreas donors. Anecdotal evidence suggests that 1/3 of a pancreas from
a living donor may produce many more viable islets than an entire
cadaveric pancreas. Basic research is needed to understand how beta cell
mass is altered after partial pancreatectomy and transplantation, the
time course, and the mechanisms for islet or beta cell rejection, loss
of function, engraftment and regeneration. Tools are needed to
distinguish among rejection, autoimmune destruction and beta cell
failure. Tools to tag islets and the technology to image them and their
function are badly needed. Especially important are large animal models
of diabetes that can be used to study transplantation.

**IV.1. Immunoisolation and Islet Immunogenicity**

> An alternative to immunosuppressive drugs could be the use of islet
> encapsulation, perhaps in alginate or behind a mechanical barrier. New
> immunoisolation devices and islet encapsulation materials are needed.
> It might be worthwhile to continue to develop these products in the
> absence of perfect materials, because devices that provide even
> imperfect immunoisolation could be very useful tools for developing
> new cell therapies in a whole animal environment, or for understanding
> the mechanisms leading to rejection or functional failure.
>
> Localized immunosuppression therapies must be developed to obviate the
> need for systemic immunosuppression. Immunogenicity should be assessed
> for alternative transplantation sites. Drug eluting materials should
> be explored to provide short term, localized immunosuppression,
> perhaps employed in islet encapsulation.
>
> It is important to understand how the islet isolation process, or
> other manipulation of islets (culture, tagging, etc.) alters their
> immunogenicity.
>
> Because the quantity of available human islets may remain limiting,
> the specific immunoisolation needs associated with xenotransplantation
> require further research.
>
> [Needed:]{.underline}

-   New immunoisolation devices, encapsulation materials and strategies;

-   Studies to determine how the islet isolation process alters
    immunogenicity;

-   Engineering methods to reduce islet immunogenicity;

-   Local immunosuppression strategies;

-   Source of abundant, high quality, relevant islet tissue for basic
    studies, such as porcine islets.

**IV.2. Islet Isolation**

> Ten Islet Cell Resource Centers (ICRs) have been established across
> the country to provide high quality human islets for treatment of type
> 1 diabetes, and for basic research. These Centers share a mission to
> improve the quantity and quality of available human islet tissue. The
> isolation process approved by the FDA cannot be altered without
> jeopardizing clinical use of the resultant islets. Therefore, although
> an important goal is to obtain enough islets to do a single-donor
> transplant, it is difficult to experiment with the isolation process
> to improve islet yield and quality. Two important barriers are
> insufficient funds for purchase of human pancreata with which to test
> new isolation procedures, and lack of criteria which would allow the
> ICRs to identify pancreata, prior to islet isolation, that would yield
> only research grade islets. Improved isolation procedures could then
> be tested on these tissues.
>
> Research is needed to identify how manipulation during the isolation
> process alters immunogenicity of the resultant islets. Animal and cell
> systems are needed to monitor this quality over time.
>
> [Needed:]{.underline}

-   Criteria to identify 'research grade' vs 'clinical grade' pancreata
    prior to islet isolation;

-   Funds to purchase pancreata for research on isolation procedures;

-   In vitro or animal-based assays are needed to test immunogenicity of
    isolated islets;

-   Tools are needed for monitoring immune processes in the transplant
    patient.

**IV.3. Islet Quality**

> "Potency" testing of islets must be established in order to predict
> transplant success. Assays to assess islet quality are needed which
> report on beta-cell-specific traits, rather than general traits like
> oxygen consumption. This is because the ratio of beta cells to acinar
> and other cells can vary greatly among preparations, and robust
> exocrine cells in a partially purified islet preparation can easily
> utilize enough oxygen to mask damaged beta cells. Candidate assays
> include apoptosis markers and measures of cell function. An adequate
> measure of potency would correlate well with the ability of the islets
> to restore insulin independence in an appropriate animal model (e.g.
> NOD-scid mice). Once such a test is established, it will be important
> to identify those factors in the islets which contribute to
> engraftment success and failure. Assessment of cadaveric vs. living
> donors is needed in order to understand the effects of brain death on
> islet quality and function.
>
> It is also important to develop means to monitor islets and their
> health, function, nutrient and oxygen levels, etc., following
> transplantation.
>
> [Needed:]{.underline}

-   New surrogate markers of islet quality, which are beta cell specific
    and correlate well with engraftment potential. This could include
    online measurement of beta cell function (e.g., ability to detect
    secreted insulin, C-peptide, zinc, other co-secreted products in
    real time);

-   Islet teams that include cell biologists should work together with
    clinicians to establish potency testing, and to identify islet
    factors important for success or failure in achieving insulin
    independence;

-   Funds to establish potency testing of human islets at all ICRs,
    especially once valid tests have been designed;

-   Studies to determine islet health following transplantation, and to
    determine those factors that contribute to success or failure in the
    host environment.

**IV.4. Agents to Improve Engraftment and Function**

> Improved knowledge of the biology of the healthy beta cell and islet
> should lead to the development of drugs and other agents that would
> promote islet function, health and engraftment in the host
> environment.
>
> [Needed:]{.underline}

-   Studies of normal biology of the human islet;

-   Pharmaceutical agents for improving islet health and function in the
    host environment.

**IV. 5. Islet Engineering**

> Genetic manipulation of isolated islets may be a promising avenue to
> create cells that have increased viability, function and engraftment
> potential. However, islets are particularly sensitive to manipulation
> and are easily damaged. New methods of altering and tagging islets are
> needed that are non-cytotoxic, that enhance or do not alter survival
> and engraftment, and that do not increase immunogenicity. Animal
> models, especially non-human primates, should be employed to test
> engineering strategies. Simple models should be used to test complex
> cell manipulations. For instance, after partial pancreatectomy,
> isolated, manipulated islets can be reimplanted in the same animal to
> study viability and function while avoiding problems associated with
> allograft rejection, autoimmune destruction, and complications of the
> diabetic environment.
>
> [Needed:]{.underline}

-   Engineered modifications or pharmaceuticals to enhance beta cell
    viability;

-   Better gene therapy tools;

-   Methods of altering islets that do not increase immunogenicity;

-   New animal models in which to test engineered islets.

**[V. Stem Cells and Cell Engineering for Beta Cell
Replacement]{.underline}**

Although much current research is focused on identifying a direct beta
cell progenitor cell, we still do not have decisive evidence that such a
cell exists in the adult. More effort is needed in engineering primary
cells, embryonic/fetal stem cells and cell lines to produce an effective
beta cell replacement. Needed are a set of criteria for "beta cell-ness"
that can be used to create, characterize and evaluate new cell
therapies. Also needed are molecules that help to induce stem cell
differentiation and replication to beta cells. The use of animal models
is to be encouraged, so that cell therapies can be developed and tested
in a whole body environment. New experimental approaches and devices may
be needed, such as an implantable isolation chamber from which cells can
be retrieved after training through interaction with a host environment.

Methods are needed for altering stem cells and cell lines to make them
invisible to a host immune system. Central to this would be an improved
understanding of those gene products that initiate or modify the self
and host immune responses.

[Needed:]{.underline}

-   Work toward making differentiated beta cells from stem cells;

-   New animal models and strategies for studying or 'training' cells in
    the environment of the whole animal;

-   Means of 'cloaking' engineered beta cells from the host immune
    system;

-   Chemical library screens to identify molecules that induce stem cell
    differentiation and replication;

-   Criteria to define "beta cell-ness".

**[VI. Beta Cell Imaging]{.underline}**

The ability to image the beta cell and its function would revolutionize
research in beta cell replacement and islet regeneration. Several
imaging projects are underway, and success will be a function of
improved imaging methods and targeting reagents. Imaging of transplanted
islets may be most feasible, whether by tagging islets prior to
transplantation or by monitoring changes in the host organ (liver).

[Needed:]{.underline}

-   Non-cytotoxic methods for tagging islets prior to transplant;

-   Imaging technologies with improved sensitivity;

-   Islet cell surface markers and highly selective targeting agents;

-   Characterization of unique physiological or functional aspects of
    islets/beta cells that could be imaged in vivo (e.g., blood flow,
    zinc secretion, enervation, etc.)

**[VII. Grant Mechanisms, NIH-Funded Consortia and Other
Infrastructure]{.underline}**

NIH has funded a variety of resources and consortia aimed at providing
different aspects of cell therapy for diabetes (appendix 1). New
projects undertaken in the cell therapy area are unlikely to require new
consortia, although collaboration among existing consortia is to be
encouraged. NIH staff are the most efficient means to coordinate among
NIH-funded consortia. At the same time, the existing consortia have
narrowly targeted goals, and should remain focused to ensure success.
They ought to be flexible enough to respond easily to good ideas and new
data, but may become less efficient if asked to take on new tasks in
translational research. Consortia can, however, be an efficient means of
distributing money for high risk translational research through Pilot &
Feasibility projects.

Basic research is likely best funded via individual regular research
grants. Although many important projects would require a team approach,
self-assembling teams might be a better solution. NIDDK could foster a
mechanism such as a database or clearinghouse to help people identify
partners with the necessary expertise.

High risk research, especially that focused on providing products, on
translation, or highly descriptive research that is not
hypothesis-driven, but that is aimed at understanding the islet, is not
easily reviewed in the same study sections as more classical R01
applications. Novel review solutions may need to be explored.

[Opportunities]{.underline}

-   Contracts administered through consortia might be used to employ
    companies to produce specific mice, antibodies and other reagents;

-   A database or clearinghouse might aid in helping investigators to
    find collaborators with the specific experience needed for a team
    approach to translational research;

-   Consortia need to be responsible for pursuing new opportunities as
    they emerge and ending projects that no longer appear promising, and
    for rewarding successful investigators;

-   Novel review solutions are needed for novel translational research
    applications.

> **List of Appendices**
>
> Appendix 1: Islet Transplantation Resources and Consortia
>
> Appendix 2: Planning Matrices
>
> The four matrices outline our past and ongoing efforts, and future
> plans in the following areas of Cell Therapy for Diabetes. These are
> arrayed as functions of time and degree of difficulty of achieving the
> stated goals.
>
> Cell-based Therapy for Insulin Delivery
>
> Non-invasive Beta Cell Imaging
>
> Human Beta Cell Regeneration
>
> Human Islet Transplantation
>
> Appendix 3: Advisors to the NIDDK / NCRR Beta Cell Working Group

**Appendix 1: Islet Transplantation Resources and Consortia**

The NIH has a comprehensive and multi-faceted approach to foster
development of islet transplantation as a cure for diabetes.

The National Center for Research Resources (NCRR) along with the NIDDK
and the Juvenile Diabetes Research Foundation International (JDRFI),
funded the **Islet Cell Resource Centers (ICR)**,
<http://www.infosci.coh.org/icr>, a consortium of ten centers whose
mission is to provide clinical-grade islets for islet transplantation;
to optimize harvest, purification, and shipment of islets while
developing tests that characterize the quality and predict the
effectiveness of transplanted islets; and to provide islets for basic
research. The Administrative and Bioinformatics Coordinating Center
(ABCC), coordinates data, requests for islets, and distribution.

The NIDDK also supports the **Collaborative Islet Transplant Registry
(CITR)**, which collects, analyzes, and disseminates comprehensive and
current data on all islet/beta cell transplants performed in the United
States and Canada (<http://spitfire.emmes.com/study/isl/index.html>).

The **Immune Tolerance Network**, funded by NIAID, NIDDK and JDRFI,
supports multi-center and single center clinical trials of islet cell
transplantation
(<http://www.immunetolerance.org/research/islet/trials.html>).

Other components of the effort to develop a cell-based therapy for
diabetes include:

> The **Non-Human Primate Islet Transplantation Consortium**
> (<http://www.niddk.nih.gov/fund/diabetesspecialfunds/consortia/NHP.pdf>);
>
> the **Beta Cell Biology Consortium**
> ([http://www.betacell.org](http://www.betacell.org/));
>
> the **Endocrine Pancreas Consortium Database**, (EPconDB,
> [www.cbil.upenn.edu/EPConDB/](http://www.cbil.upenn.edu/EPConDB/))
> which developed and distributes pancreas-specific cDNA microarray
> chips for human and mouse;
>
> and the **Type 1** **Diabetes Rapid Access to Interventional
> Development** (T1D-RAID)
> (<http://www.niddk.nih.gov/fund/diabetesspecialfunds/T1D-RAID/> ).

Appendix 2: Planning Matrices

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**Appendix 3: Advisors to the NIDDK / NCRR Beta Cell Working Group**

Douglas A. Melton, Ph.D.

Professor, Department of Molecular and Cell Biology

Harvard University

Camillo Ricordi, M.D.

University of Miami School of Medicine

Surgery/Diabetes Research Institute

John W. Turk, M.D., Ph.D.

Professor, Department of Medicine

Washington University School of Medicine

David Harlan, M.D.

Chief, Transplantation and Autoimmunity Branch

NIDDK-NIH