2012 — 2016 |
Bhattacharjee, Arindam |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ionic Mechanisms Underlying Dorsal Root Ganglion Excitability @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): Pain sensation in neuropathic pain is complex consisting of weakness, sensory deficits and numbness, reflex changes, abnormal sensations that occur either spontaneously or in reaction to external stimuli, hyperalgesia and allodynia. Perturbations in dorsal root ganglion (DRG) neuron excitability are key in precipitating neuropathic pain, especially during diabetes, the most common cause of neuropathic pain. During diabetes, the p38 mitogen-activated protein kinase (p38MAPK) signaling system is activated and when this pathway is inhibited, diabetes-induced neuropathic pain is attenuated. However, the major ion conductances involved in the neuropathic process of DRG neurons are unclear. DRG neurons possess high levels of a novel, understudied family of potassium channels called sodium-activated potassium channels (KNa). Our previous work has shown that KNa is a considerable component of the outward potassium current and is responsible for firing accommodation in DRG neurons. When we experimentally reduce the expression of these channels in DRG neurons, it produces hyperexcitability that resembles neuropathic neurons. There are two genes encoding these channels, Slack and Slick. In heterologous expression systems, the Slick and Slack subunits can co-assemble to form heteromeric channels systems with very slow activation kinetics ideal for controlling firing accommodation. Moreover, homomeric Slick channels appear to be subject to Nedd4l-dependent ubiquitination, suggesting that Slack/Slick heteromeric channels are the preferred configuration of native KNa channels. Slack and Slick also have p38MAPK consensus phosphorylation sites proximal to the sodium binding/gating region of the channels. A decrease in KNa channel activity likely ensues after diabetes-activated p38MAPK signaling. Since diabetes also affects transcriptional activities, we expect to find long-term changes in KNa channel expression in neurons. Using electrophysiological, biochemical, molecular, pain behavioral assays and a previously uncharacterized Slick knockout mouse, we will test the hypotheses: heteromeric KNa channels constrain sensory neuron hyperexcitability and neuropathic pain is associated with decreased KNa channel activity in DRG neurons. The specific aims are (1) To study the regulation of DRG KNa channels by p38MAPK (2) To investigate the subunit properties of KNa channels in DRG neurons (3) To study neuronal KNa channel activity during diabetes and compare pain behavior to Slick knockout mice. This research project will assess the involvement of KNa channels in the diabetic neuropathy.
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0.954 |
2018 — 2020 |
Bhattacharjee, Arindam |
R44Activity Code Description: To support in - depth development of R&D ideas whose feasibility has been established in Phase I and which are likely to result in commercial products or services. SBIR Phase II are considered 'Fast-Track' and do not require National Council Review. |
A Comprehensive Newborn Screening Solution For Duchenne and Congenital Muscular Dystrophies
ABSTRACT A Comprehensive Newborn Screening Solution for Duchenne and Congenital Muscular Dystrophies (Fast Track SBIR) Congenital genetic abnormalities are a leading cause of childhood mortality and morbidity. While routine newborn screening (NBS) has dramatically improved health outcomes, many congenital disorders such as Duchenne muscular dystrophy (DMD) and other congenital muscular dystrophies (CMD) are not currently detected by routine NBS. Presymptomatic identification through NBS is critical to facilitate earlier initiation of therapies and for improved long-term outcomes of patients with DMD or other CMDs. With several new therapies on the horizon, the interest in DMD screening has grown considerably and there is reason to believe testing may be adopted for public health screening in the next five years. The goal of this Fast Track SBIR project is to develop a complete testing solution for efficient newborn screening of DMD and CMDs from dried blood spot (DBS) specimens. The system will consist of automated, low volume biochemical assays for creatine kinase (CK) enzyme activity and CK isoform expression (CK-MM and CK-MB) followed by 2nd-tier targeted next generation sequencing (tNGS) in CK (+) individuals to detect common casual gene variants associated with DMD and CMDs. The proposed 1st-tier biochemical tests will leverage Baebies' proprietary SEEKERTM platform, which is FDA cleared for NBS of lysosomal storage disorders, to provide high throughput, multi-analyte CK testing to the newborn screening market. The biochemical measurements will be combined into a decision algorithm to reduce false positives and eliminate false negatives. A similar strategy is used successfully in some state NBS programs for detection of thyroid conditions, where TSH and T4 are measured simultaneously and correlated to better define disease state. The addition of tNGS for 2nd-tier analysis will provide further precision to our system and has the potential to revolutionize the care of infants and young children with elevated CK levels. After initial validation of the system, the next steps will be to extend the clinical portion of the study to generate evidence for nomination to the Recommended Universal Screening Panel (RUSP), which states use to inform their NBS offerings. We anticipate further validation within a Phase IIB trial concomitant with seeking CLIA certification for the screening service and eventually FDA approval of the biochemical test system. The final product of this research will be differentiated from competing single analyte CK tests for NBS by its use of an automated, multianalyte biochemical assay (at minimal added cost compared to single analyte tests due to the tiny reagent volumes required) and the rapid, small sample tNGS workflow ? which combined will enable efficient identification of newborns at risk for DMD/CMDs with lower false positive rates and no false negatives.
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0.91 |
2018 |
Bhattacharjee, Arindam |
R43Activity Code Description: To support projects, limited in time and amount, to establish the technical merit and feasibility of R&D ideas which may ultimately lead to a commercial product(s) or service(s). |
Feasibility and Validation of An Integrated Newborn Screening Algorithm With the Targeted Next Generation Sequencing (Tngs) Technology as Part of a 2nd Tier Test For Pompe and Mps I
ABSTRACT Due to the lack of widespread 2nd-tier screening tests, newborn screening providers and parents struggle to understand the ramification of genetic conditions detected during the neonatal period. This is especially true in newborn screening for Lysosomal Storage Diseases (LSDs) such as Pompe and Mucopolysaccharidosis Type I (MPS I), where pseudodeficient variants result in a lack of enzyme activity in the newborn dried blood spot (DBS) screening test, but present with normal endogenous enzyme activity and with no overt disease manifestations. Current screening algorithms do not routinely identify or provide molecular genetic etiology for LSDs because the genotype is not routinely monitored. However, frequency of pseudodeficiency mutations can be as high as 4% in certain populations and can be a significant source of false positive (+) results. Enhanced sensitivity and specificity in newborn screening for Pompe and MPS I is essential, but extremely challenging. Correct diagnosis of a disease, and its underlying biochemical and molecular basis, is not only critical for successful differential diagnosis and treatment early in life but it is also important in reducing anxiety in parents and unnecessary burdens on follow-up programs. Current gene sequencing methods including genome scale sequencing [Whole Exome and Whole Genome (WES and WGS, respectively)] used for confirmatory diagnosis are impractical in newborns and do not scale. A targeted next-generation sequencing (tNGS) panel method to address deficiencies in current testing can be used as a routine 2nd-tier newborn screen to significantly improve sensitivity and specificity. The panel designs of single genes are impractical for scaling in screening conditions due to the rare nature of LSDs and maintenance of multiple assays. Genome scale sequencing approaches are not comprehensive across non coding regions (introns, promoters, etc.) with gaps in coverage or high costs that are prohibitive for screening paradigms. We will create a comprehensive newborn specific gene panel for LSDs by establishing sample collection and processing workflows more appropriate for newborns. We will demonstrate the power of tNGS for reducing false (+) results by identifying clinical variants and pseudodeficiency mutations in Pompe and MPS I. Our goal is to develop an assay with high sensitivity and specificity. We will demonstrate the value of the assay when used as a 2nd-tier screen following 1st-tier enzyme analysis via the Baebies' FDA cleared SEEKER device for high throughput newborn screening of LSDs. We will use the results to develop an algorithm that can be used by newborn screening programs for accurate interpretation of screening results. These studies will eventually allow CLIA/CAP validation of our tNGS methodology and permit us to offer our screening panel for LSDs on a commercial basis. Our approach has the potential to rapidly and simultaneously screen for hundreds of other LSD or newborn conditions with future development. Early identification of many of these additional disorders is critical for rapid and appropriate management.
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0.91 |
2019 |
Bhattacharjee, Arindam |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Disrupting Ion Channel Scaffolding to Treat Neuropathic Pain @ State University of New York At Buffalo
Project Summary Millions of people suffer from neuropathic pain and the current treatment protocols and therapies are limited in their effectiveness. There is a clear need for the development of novel, non-addictive treatment strategies for neuropathic pain. Dorsal root ganglion (DRG) neuronal hyperexcitability is central to the pathology of neuropathic pain, so much so that application of local anesthetics is currently considered as a first line treatment strategy for neuropathic pain. However, studies on the efficacy to these local anesthetic patches has been mixed. Notwithstanding, the coordinated movement of ion channels, especially voltage-dependent sodium channels, from intracellular pools to the sites of nerve injury has been suggested to be the underlying cause of electrogenesis and ectopic firing in neuropathic pain. Thus instead of blocking sodium channels, an alternative method to treating neuropathic pain could be to disrupt their trafficking. Recent studies have indicated that WW domain-containing ubiquitin ligases are downregulated during nerve injury causing an increase in membrane targeting of sodium channels. The scaffold proteins responsible for sodium channel targeting and membrane stabilization in DRG neurons have not been fully characterized although our recently published studies identified the Magi1 scaffold protein a potential candidate. Using electrophysiological, biochemical, molecular, pain behavioral assays, and a novel in vivo method to knockdown genes in DRG neurons, we will test the hypotheses: MAGI1, a PDZ and WW domain-containing scaffold protein, is critical for NaV membrane expression in rodent and human DRG neurons and targetable for the treatment of neuropathic pain. The specific aims are: (1) To study the effects of Magi1/MAGI1 knockdown on rodent and human DRG neuronal excitability and to determine if Magi1 deficiency impacts the development of neuropathic pain during nerve injury. (2) To investigate how Magi1 is regulated by neuropathic pain and whether downregulating Magi1 can provide analgesia after established neuropathic pain. (3) To demonstrate that local application of WW domain peptidomimetics can reduce neuropathic pain behavior. This research project will validate MAGI1 as a therapeutic target to treat neuropathic pain.
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0.954 |
2019 — 2020 |
Bhattacharjee, Arindam |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
The Role of the Ap2 Adaptor Complex in Inflammatory Pain @ State University of New York At Buffalo
Project Summary One of the cardinal features of inflammatory states is that normally innocuous stimuli produce pain. Current pain-relieving drugs include nonsteroidal anti-inflammatory drugs, which are aimed at the interdiction of prostaglandin production, corticosteroids and opioids. However, the side effects and some cases addiction potential associated with these drugs limit their long-term use especially during chronic inflammatory pain. To develop novel, non-addictive analgesics, there remains an urgent need to understand how inflammation produces the change in nociceptor firing that underlies pain perception. In this proposal, we aim to provide proof of principal that in dorsal root ganglion (DRG) neurons, adaptin 2 clathrin-mediated endocytosis (AP2- CME) is a principal facilitator of inflammatory-induced nociceptor sensitization. We have previously demonstrated that in response to protein kinase A (PKA) stimulation, Slack KNa channels are internalized via AP2-CME from DRG neuronal membranes and this caused hyperexcitability. Furthermore we showed that inhibiting AP2-CME prevented PKA-induced neuronal hyperexcitability. Preliminary studies now indicate that in vivo knockdown of the AP2 alpha subunit AP2A2 specifically within DRG neurons, substantially reduces inflammatory pain behavior. Here, we will apply a combination of protein chemistry, immunohistochemistry, electrophysiology, spinal cord physiology, pain behavior assays and a novel in vivo gene knockdown approach to test the hypothesis that AP2-CME is a key regulator of nociceptor sensitization. The specific aims are 1) to determine whether AP2-CME controls basal excitability and neurotransmission 2) To demonstrate that reducing AP2-CME mitigates inflammatory pain. This research project will reveal the central role AP2-CME plays in pain signaling.
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0.954 |
2020 |
Bhattacharjee, Arindam |
R44Activity Code Description: To support in - depth development of R&D ideas whose feasibility has been established in Phase I and which are likely to result in commercial products or services. SBIR Phase II are considered 'Fast-Track' and do not require National Council Review. |
Feasibility and Validation of An Integrated Newborn Screening Algorithm With Targeted Next Generation Sequencing (Tngs) Technology as Part of a 2nd-Tier Test For Pompe and Mps I
ABSTRACT Feasibility and validation of an integrated newborn screening algorithm with targeted Next Generation Sequencing (tNGS) technology as part of a 2nd-tier test for Pompe and MPS I Newborn screening (NBS) utilizes high throughput primary (1st-tier) screening assays paired with referral and clinical follow-up testing to identify babies who are affected with certain inherited disorders. Where necessary, 2nd-tier tests are performed prior to referral for follow-up testing in order to reduce the number of false positives (screen positive samples that are determined to be unaffected). False-positive newborn screens have undesired consequences for both families and the public health lab referral system, including: high cost associated with additional confirmatory testing, extra testing burden on referral centers, parental anxiety, parent-baby bonding issues, and added stress to the baby with additional tests and blood draws. There exists a strong need to reduce the rate of false positive newborn screens by implementing 2nd-tier molecular or biochemical tests prior to referral. This Phase II project will be a continuation of our successful Phase I research, which generated an integrated 2nd-tier targeted next generation sequencing (tNGS) workflow capable of identifying both point mutations and large deletions/duplication events from dried blood spot (DBS) specimens. We will continue to focus on Pompe disease and Mucopolysaccharidosis Type I (MPS I) -- two lysosomal storage disorders that were recently recommended for universal NBS in the U.S., but have been challenging to implement as NBS tests due to the high rates of pseudodeficient variants. Currently, 2nd-tier testing via either additional biochemical analysis or gene sequencing for known pathogenic variants are used to identify pseudodeficiency and reduce false positive test rates. We will expand our novel tNGS 2nd-tier workflow by: 1) developing bioinformatic tools for variants of uncertain significance (VUS) cut-off and cross-reactive immunological material (CRIM) status prediction; 2) improving our existing copy number variability (CNV) caller; and 3) integrating additional enzyme measurements and demographic data with the tNGS score. Demographic data has previously been shown to correlate to measured enzyme activities due to biological factors and DBS sample variability. Our algorithm will provide a better disease state call and associated data for improved follow-up care, provide critical predictions for disease onset and treatment considerations. The 2nd-tier tests developed through this work will be sold initially as a diagnostic send-out service and eventually as kits to public health labs that are currently screening, or planning to screen for Pompe disease and MPS I. Affected individuals who are identified using our tests will be referred to follow-up earlier and will have an accelerated path to disease confirmation and treatment. These features are especially important for Pompe disease, where a delay in therapeutic intervention of just days is known to negatively impact long term health outcomes. The approaches developed through this work have the potential to be expanded to cover dozens of enzyme deficiencies from the same primary dried blood spot sample.
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0.91 |