ADA2 Diagnostic Platform

Deficiency of Adenosine Deaminase 2 (DADA2) is a relatively new (identified in 2014) genetic disorder of immunity (inborn error of immunity) with autosomal recessive inheritance due to pathogenic variants in the ADA2 gene resulting in loss-of-function (LOF) of the ADA2 protein, an enzyme critical for normal immune and vascular function (Zhou Q, et al. Early-Onset Stroke and Vasculopathy Associated with Mutations in ADA2. New Engl J Medicine. 2014) & (Elkan PN, et al. Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy. New Engl J Medicine. 2014). The clinical phenotype of DADA2 is characterized by autoinflammation causing an early-onset vasculopathy, immunodeficiency, bone marrow failure and other organ dysfunction. DADA2 has been diagnosed in both children and adults. The disease has variable penetrance with a mortality rate between 8% and 10%. Most patients develop symptoms before 16 years of age. ADA2 enzyme testing is currently not part of the national newborn screening program.

Early diagnosis of the disease will lead to early intervention to prevent and/or reduce the morbidity and mortality associated with the disease. Quantitation of ADA2 enzyme levels in blood is an effective approach for the rapid diagnosis of DADA2. Diagnosis of the disease can ensure early access to treatment with TNF alpha-blockade using anti-TNF alpha inhibitor therapies. This is currently the most effective therapy for stroke prevention in these patients. We propose to create a new and rapid approach to diagnosis of DADA2 using our “ADA2 Diagnostic Platform®” technology (capillary tubes, test strips, and hand-held meter PixoTest® (iXensor)), which will enable physicians to accurately identify patients and carriers based on ADA2 enzyme levels. The benefits of this novel approach are:

• Early detection leading to reducing healthcare costs, and lower morbidity and mortality from late diagnosis causing a higher incidence of strokes and bone marrow failure, requiring more aggressive intervention, including hematopoietic stem cell transplantation.
• An innovation with global impact, enabling populations around the world access to an inexpensive screening method improving quality-of-life (QOL) and minimizing the diagnostic odyssey.
• It will offer a paradigm that could potentially be replicated for the diagnosis of other rare genetic diseases, which may be amenable to utilization of point-of-care testing. 

Our solution utilizes a novel enzyme-detection assay which is performed on a colorimetric diagnostic platform with a portable and affordable test strip technology impregnated with reagents for quantitation of ADA2 enzyme activity in human blood from a finger or heel-stick. Blood is collected via a fixed volume capillary tube and dispensed in the sample well. Blood flows vertically, where a primary blood separation membrane is impregnated with agglutinating agents, and size-excludes red (RBCs) and white (WBCs) blood cells away from the plasma. The secondary blood separation membrane (3 um) filters out any remaining cells. Plasma reaches the reagent layer, which is embedded with an indicator, bioactive components, and a substrate that specifically targets ADA2 enzyme activity (Units/L). The test strip is inserted in an FDA-approved PixoTest® (iXensor) ® device for the quantitation of ADA2 enzyme activity, where end-color (RGB values) is directly proportional to the plasma ADA2 enzyme concentration. The incorporation of a selective inhibitor (EHNA, erthyro-9-(2-hydroxy-3-nonyl) adenine) of the other major isoform of ADA, ADA1, ensures specific and accurate detection of ADA2 for the diagnosis of DADA2. Clinical validation of the assay will be performed using ADA2 patient and carrier samples, as well as healthy controls provided by Dr. Pui Lee, a pediatric rheumatologist at Boston Children’s Hospital, and an expert in DADA2.

We have already generated preliminary data using such samples in a commercial ADA2 enzyme assay (Diazyme). We have also generated additional data demonstrating a robust dose-response curve of ADA2 enzyme concentration versus RGB values with these patient plasma samples as proof-of-principle. We will obtain de-identified patient, carrier, and healthy control plasma and/or serum samples from various individual investigator-generated biorepositories nationally for additional analytical validation. 

This novel point-of-care testing (POCT) diagnostic device (ADA2 Diagnostic Platform) will facilitate early diagnosis of patients with a rare disease causing early-onset strokes and have a dramatic impact on prognosis and outcomes in the DADA2 patient population.

DADA2 likely has a higher prevalence than currently diagnosed. Only 400 patients are presently reported in the medical literature. Recent functional studies by Dr. Pui Lee and colleagues at the Broad Institute/MIT suggest that genetic ADA2 variants in public databases combined with the known ADA2 variants that cause DADA2 show that the carrier frequency of DADA2 is approximately 1 in 236 individuals, corresponding to an expected DADA2 disease prevalence of 1 in 200,000 individuals (Jee H, et al. Comprehensive analysis of ADA2 genetic variants and estimation of carrier frequency driven by a function-based approach. J Allergy Clin Immun. 2021). This data posits that DADA2 will likely be one of the more common among the rare diseases, emphasizing the importance of this project.

Healthcare access inequity may result in further delays in diagnosis, especially in low-income families, who do not have access to medical insurance, optimal treatment, genetic counseling (https://www.ncbi.nlm.nih.gov/books/NBK538442/), and other necessary benefits.

Further, as per the 2020 U.S. Census, the rate of uninsured children is increasing, especially among those at or below 139% of the poverty line. Access to medical facilities, such as emergency rooms (ER) where POCT devices are deployed, may eliminate or redress some of the healthcare gaps.

Early diagnosis of DADA2 has practical clinical implications, especially from the standpoint of stroke management. While patients presenting with features of a stroke are normally considered for immediate anticoagulation, this approach may cause paradoxical complications in DADA2, exacerbating the complications and increasing the risk of hemorrhagic stroke (Barron KS, et al. The Spectrum of the Deficiency of Adenosine Deaminase 2: An Observational Analysis of a 60 Patient Cohort. Front Immunol. 2022). Patients presenting with strokes due to DADA2 deficiency require immediate management with anti-TNF alpha inhibitors, rather than anticoagulants. Finally, the clinical heterogeneity of DADA2 means that these patients may present with different phenotypes to different clinical sub-specialties (hematology, rheumatology, dermatology, neurology, ophthalmology, immunology etc.), and a POCT device could be easily made available in these various areas. This will facilitate the consideration of DADA2 in the differential diagnosis of patients who present with the constellation of symptoms associated with this disease, and enable access to immediate screening, with additional confirmatory testing ordered as needed, based on this result.

The mission of IVDS is to develop simple, low-cost, quantitative POC assays/tests for novel biomarkers and rare diseases. The Co-PI Robert Harper (RH) has significant expertise in developing such assays for the inborn errors of metabolism (IEM), and it is a logical extension to develop similar tests for the inborn errors of immunity (IEI), such as DADA2. RH has 16 successful Small Business Innovation Research (SBIR) awards related to the IEM. RH also holds 6 patents and has more than 25 years of experience in POC assay development. He was instrumental in developing the first FDA-approved home test for measuring total cholesterol, (ENACT, ActiMed Laboratories). His work spans global collaboration for the development of FDA-approved diagnostic devices with EKF Diagnostics, Lycotek, Polymer Technology Systems, Aspen Laboratories and PortaScience.

The other, Co-PI Chip Chambers (CC), is a former Assistant Professor of Surgery, Chief of Endocrine Surgery, and Director of the Endocrine Surgery Center at Vanderbilt University Medical Center in Nashville, Tennessee. He currently holds an emeritus academic rank at the University. CC is the Founder and President of the DADA2 Foundation, the only non-profit organization dedicated to this disease (www.dada2.org). The DADA2 Foundation was started in 2016 by CC and his family in honor of their two children affected by this disease. One of the missions of the DADA2 Foundation is to form productive and innovative partnerships between clinicians, researchers, biotech, pharma, and patients in the interest of diagnosing and curing this disease. CC is a relentless advocate for DADA2 patients and is the co-author of several scientific papers on DADA2. In recognition of his work on rare diseases, CC has been appointed to the newly created Rare Disease Advisory Council by the Governor of Tennessee.

Since its inception in 2016, the DADA2 Foundation has hosted 3 international scientific conferences and 3 patient-centered gatherings, reaching 500+ researchers and 250+ patients in over 39 countries. The DADA2 Foundation has been closely engaged with the scientific community who have published over 150 papers on DADA2 in the past eight years. 

We believe that all patients deserve maximal access to the best treatment available for their disease, once diagnosed. However, we know that this paradigm does not exist in reality. DADA2 is not a disease restricted to the developed world. It is a global disease, and one of 7,000 rare diseases affecting approximately 300 million people worldwide. It is far more challenging to solve the problem of all 7,000 rare diseases, so we have selected one rare disease to tackle, DADA2. We want to ensure that every one of the potential 37,000 predicted patients with DADA2 have access to early diagnosis, and appropriate treatment. To ensure the latter, we have to first achieve the former. This proposal seeks to precisely do that; diagnose DADA2 patients early. We believe that a partnership with and support from MIT Solve will allow us to reach our goal of making DADA2 diagnostic testing accessible and affordable across the globe.

Genetic testing using next-generation sequencing (NGS) is the gold standard for identification of pathogenic variants in the ADA2 gene. However, genetic testing alone is insufficient for diagnosis in many patients due to novel variants or variants of uncertain significance (VUS). Therefore, corroboration of genetic data with protein studies is often essential for a complete diagnosis. Currently, there are only three research laboratories in the country that perform ADA2 enzyme quantitation. There is no routine clinical diagnostic test meeting the standards of CLIA and the College of American Pathologists (CAP), the regulatory body overseeing diagnostic testing in the U.S. on behalf of the Center for Medicare and Medicaid Services (CMS). Genetic testing has become cheaper over the years, but it is still inaccessible for many in developing countries. Also, it still takes 2-3 weeks (targeted panels) to a few months (exome/genome) to obtain genetic data, in which time period a patient may not be diagnosed or treated and may suffer a catastrophic clinical event. The proposed POCT offers a novel solution to the problem of early diagnosis of DADA2, while simultaneously addressing both accessibility and equity. Individuals with absent ADA2 enzyme activity are exceedingly likely to have DADA2. Quantitation of ADA2 enzyme levels in a regular research or clinical laboratory requires more elaborate methodology with associated costs of instrumentation, personnel, and reagents. The POCT approach obviates the need for elaborate infrastructure for screening purposes. We recognize that many POCT assays require confirmation with more traditional validated methods, but even identification of patients by a screening test significantly reduces time and cost of diagnosis and makes it available to a larger fraction of the population. Thus, the ADA2 Diagnostic Platform offers a more portable, cost-effective approach for quantitation of ADA2 enzyme levels and diagnosis of DADA2. Our method utilizes a proven test strip platform for rapid, quantitative, colorimetric detection of this specific analyte, ADA2, in whole blood. Currently, there are no POCTs for inborn errors of immunity, and thus developing this platform for DADA2 diagnosis will expand the diagnostic horizon for these rare diseases and have a profound impact on clinical practice for this and similar diseases globally.

Our solution will have a significant impact in the rare disease space, especially for DADA2, by bringing an affordable and rapid POCT device to patients worldwide and encouraging understanding, collaboration, and early treatment for DADA2. During the next year, the DADA2 Foundation will continue to host international scientific conferences, expand patient-centered gatherings, and to engage the scientific community to increase awareness of DADA2. In parallel, IVDS will optimize the ADA2 Diagnostic Platform to ensure sufficient resolution between ADA2 deficient, ADA2 carriers, and ADA2 normal patients. The verification and validation of this platform will be completed by year 2. The following years will be focused on procuring funding for submission of the FDA Pre-IDE submission and 510 K submission, while seeking collaboration with pharma companies for dissemination of the platform. By year 5, we hope that worldwide dissemination is achieved and the impact on patients’ lives is made clear.

The development and dissemination of the ADA2 Diagnostic Platform is well-aligned with the UN Sustainable Development Goals SDG 3 by “Ensuring healthy lives and promoting well-being” by developing cost-effective treatments and products.

In year 1 we will complete the development work:

• IVDS will finalize the formulation for the ADA2 reagent membrane to provide resolution between ADA2 deficient <5 Units/L activity, ADA2 Carrier, 5-10 units/L activity and ADA2 normal > 10 Units/L activity. IVDS will run verification and validation testing for the ADA2 reagent membrane and blood separation membranes.
• IVDS will utilize the Clinical and Laboratory Standards Institute (CLSI) approach on the continued performance testing test to measure our success.

• Reference interval in a normal healthy population (CLSI C28-A3c)               I. Test results obtained from 120 apparently healthy individuals (60 males + 60 females) without the disease or deficiency in question; the 2.5th percentile of the distribution of test measurements will serve as the one-sided lower reference limit for the test.

• Within-laboratory precision (CLSI EP05-A3)

      I. Test 3 or 4 specimens with varying concentrations; should have one specimen at or close to each relevant clinical decision point (or cutoff), and one specimen in the upper half of the measuring range

      II. Coefficient of variation (%CV, i.e., standard deviation as a percentage of the mean) should be ≤ 5% near all clinical cutoffs.

• Linearity (CLSI EP06-A)

       I. Study performed using two whole blood specimens, one with concentration below the anticipated LoQ and one with concentration above the anticipated upper limit of the measuring range

       II. Specimens are mixed in various ratios to obtain 7-11 intermediate levels with concentrations spanning the range

      III. Each level is tested in 3 to 5 replicates

       IV. Regression analyses performed to determine whether data is linear, or whether the deviation from linearity (when fitting higher-order non-linear models) is within specifications

       V. Deviation from linearity should be within ±10% for all levels within the measuring range of the test.

        VI. Protocol for linearity validation. 

 IVDS, Dr. Pui Lee and the DADA2 Foundation will have biweekly meetings to discuss results and engage other researchers when needed. CC will explore leveraging individual investigators for the dissemination of the ADA2 Diagnostic Platform as well as work to create additional biorepositories at academic medical centers and other professional organizations who share a common mission. The goal will be to network to support the funding for the validation of the ADA2 Diagnostic Platform, expand the awareness of clinicians and understanding of this IEM. and procure additional funding for the dissemination of the platform to emergency rooms (ER), first in the U.S. and then globally. Following a rollout in the ERs, we will then work on placing the device in many of the sub-specialists’ offices that might see a DADA2 patient.

Our solution will provide a simple and user-friendly method of determining ADA2 enzyme activity in blood samples using a rapid point-of-care testing platform. This will assist physicians with diagnosing and treating patients by supplementing DADA2 detection in patients with known or likely genetic mutations of the ADA2 gene and by providing a diagnostic checkpoint for patients with unknown genetic profiles but who express relevant phenotypic conditions. This will directly lead to early therapeutic intervention options and significantly improve patient prognosis. In the long-term, a rapid point-of-care diagnostic for DADA2 will improve our understanding of the symptoms of this disease, diagnose patients that go untested, and understand how DADA2 may relate to patient populations while encouraging collaborative efforts in the search for a cure for DADA2.

The ADA2 Diagnostic Platform solution is driven by our expertise in dry-phase chemistry and the integration of test strips capable of separating plasma from whole blood and a patented, hand-held, image analyzing meter, for correlating end-color with a colorimetric change driven by the presence of the ADA2 enzyme. The hand-held meter PixoTest® (iXensor) ® that collects, analyzes, and stores data for sharing and analysis will supplement the diagnostic strips and allows for a mobile and versatile platform for testing blood concentrations of the ADA2 enzyme, diagnosing DADA2 patients and carriers, and managing patient data. This data would also augment a Natural History Study and Patient Registry that is under development by the DADA2 Foundation.

The IVDS team is well diversified team and all inclusive. IVDS has weekly meetings to brainstorm projects and innovative ideas for the development of novel assays. The scientific team consists of Hannah Slage (Pakistani), Tom Christiani Ph.D. (U.S.), M. D. Kabir, Ph.D. (Bengali) Robert Mosley Ph.D. (U.S.), George Temeng MSc (Ghanaian).

Similarly, the DADA2 Foundation recognizes that DADA2 is a global disease. Research today shows few geographical, ethnic, gender, or racial boundaries. A single clinician-researcher, Pui Lee, M.D., Ph.D. at Boston Children’s Hospital is seeing African American, Hispanic, Middle Eastern, and Caucasian patients in his cohort of 12 patients.

Today, DADA2’s research team is diverse, including:

• Primary study sites in Boston and San Raffaele in collaboration with some of the largest DADA2 patient cohorts in the world including the NIH (U.S.A.), Turkey and India.
• The PIs of a newly proposes study were born in China, Italy, Mexico, and U.S. Two of the PIs are female and two are early-stage investigators. Females represent at least 50% of each lab and underrepresented minority groups are also well represented.
• Patient conferences engaging 250+ patients/families from more than 20 countries, conducted with translators for those not speaking any English. Additionally, organically driven patient/physician gatherings take place in Netherlands, India, and United States.
• International conferences that, most recently in 2020, included 500 clinicians and researchers, representing > 39 countries. More than 50% of our presenters were female.
• The DADA2 Foundation’s staff, intern, and board that represents a balance of men and women in leadership and research, global countries from Africa to the U.S., diverse sexual orientation, and families created through adoption.
• Ongoing DEI training through resources provided by the Chan Zuckerberg Initiative (CZI) capacity building resources. 

The DADA2 Foundation or its subsidiaries will partner Jonathan Chapman, CEO of Polymer Technology Systems (PTS), for the marketing and dissemination of the ADA2 Diagnostic Platform. Polymer Technology Systems is in the business of supplying the highest quality point-of-care diagnostic products to the global health care market. PTS currently has the infrastructure and channels of distribution. Their products are represented in more than 100 countries worldwide some of which are underserved populations.

It is anticipated that in Year-1, the DADA2 Foundation will fund the completion of the development work and validation. We anticipate by Year-5 the ADA2 Diagnostic Platform will generate over 9 million dollars. Much funding will be recycled back into the program to expand the clinicians’ awareness of DADA2 as a newly diagnosed disease, outline testing features, and the diagnosis of the patients and their families for carriers of the DADA2 gene and treatment prior to physiological manifestations. In Year-2 the DADA2 Foundation will submit a Pre-IDE to the FDA to determine the precise regulatory pathway for Denova submission.

As well, DADA2 Foundation will procure additional funding for Year-2 to:

• Task 1.0 Optimize the Pixo Test Meter Algorithm for Patient i.d., Curve set and lot expiry.
• Task 2.0 System Case, Packaging, and Delivery Logistics
• Task 4.0 System-Level Technical Verification and Usability Testing
• Task 5.0 Market Assessment and Roadmap
• Task 6.0 Domestic, International, and FDA Regulatory Compliance
• Submission to the U.S. FDA for market clearance of the ADA2 Diagnostic Platform

The main source of revenue will be from selling the ADA2 Diagnostic Platform (meter and test strips) directly to hospitals. As there is currently no POCT for DADA2 on the market, we anticipate that we can capture a significant share of the market potentially selling to over 70% of U.S. hospitals by year 5. Based on cost estimates this will result in over $9 million in profit by year 5. Additionally, we are continually open to further partnerships, licensing opportunities, and grant funding.

1. IVDS, founded in 2017, has received over 7 million dollars in funding from the National Institutes of Health and National Science Foundation specifically to develop quantitative Point-of-Care Tests for diagnosis and monitoring of the Inborn Errors of Metabolism. IVDS has incorporated a coating system, slitter to coat and slit reagent membrane, and blood separation membranes. As well we have implemented a dehumidification system under < 25% RH to manufacture test strips. This is all done with SOPS under a Quality Management System using Medical Device Software via Green Light Guru. https://www.greenlight.guru.
2. Our intention will be to license or sell this technology to some of the companies with the infrastructure and channels of distribution to disseminate the test strip and meters to hospitals and clinicians. As well, the DADA2 Foundation has global reach and is well connected to established DADA2 clinics and potential clinics. The DADA2 Foundation will seek investment and licensing opportunities from Janssen Biotech, Inc., and Novartis, the leading manufacturers and distributors of TNF alpha inhibitors. They would be keen on having a POCT to diagnosis DADA2 and use their drugs in the treatment of the disease.