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David Fruman, PhD »

“mTOR Kinase Inhibitors: Cancer Therapeutics Repurposed for Treatment of Autoimmune Disease”

Specific Aims and Research Plan:

Rapamycin (RAP) is an immunosuppressive drug used to prevent organ rejection. RAP is a partialinhibitor of the cellular kinase mTOR. A novel class of mTOR kinase inhibitors (TOR-KIs) block mTORactivity through a distinct biochemical mechanism. TOR-KIs have much greater anti-cancer activity thanRAP in preclinical tumor models, and have entered clinical trials for oncology. However, the potentialapplication of TOR-KIs for autoimmune disease has not been addressed. TOR-KIs achieve anti-canceractivity at doses that are well tolerated and do not cause generalized immunosuppression. Preliminary dataindicate that TOR-KIs selectively suppress autoimmunity without impairing host defense. Therefore, TORKIshold promise as novel treatments for multiple sclerosis. In this project we will use an experimentalautoimmune encephalomyelitis (EAE) model of MS in mice to test the efficacy of TOR-KIs, while testingsafety using a model in which mice are infected with a virus in the central nervous system.The objective is to test the hypothesis that TOR-KIs can reduce the incidence and severity of EAE whilepreserving anti-viral immunity. We will use TOR-KI compounds that are currently in clinical trials foroncology, are known to be well tolerated in mice at doses with anti-cancer activity, and are available forpurchase from commercial vendors.

Using these compounds we will address two specific aims:1. Determine whether TOR-KI treatment reduces the incidence and severity of EAE in mice. We willcollaborate with Dr. Craig Walsh, whose lab has experience with the myelin oligodendrocyteglycoprotein (MOG) model of mouse EAE. We will evaluate the disease course in mice treated withtitrated doses of TOR-KIs, and will determine the mechanism of protection at the cellular level.Specifically, we will test the hypothesis that TOR-KIs increase the generation of regulatory T cells(Tregs) and promote the retention of activated T cells in secondary lymphoid tissues.2. Determine whether TOR-KI treatment impairs the immune response to a neurotropic virus. A majorobstacle for immunosuppressive therapy in MS is the potential for emergence of viral infections in theCNS. We will collaborate with Dr. Thomas Lane to determine how TOR-KIs affect the response to aneurotropic strain of mouse hepatitis virus (MHV). Preliminary data indicate that AZD8055 treatmentdoes not impair the clearance of MHV from the central nervous system. We will repeat and expandthese studies to compare parameters of immune activation in mice treated with TOR-KIs compared tomice treated with vehicle alone.Innovation and Translational PotentialThis proposal is innovative because we are “re-purposing” an existing class of experimental drugs for aclinical application that is fundamentally different. TOR kinase inhibitors (TOR-KIs) have been developedspecifically for use as cancer therapeutics. The goal of this study is to test the innovative hypothesis thatTOR-KIs can oppose autoimmunity and ameliorate the disease course in a mouse model of multiplesclerosis (MS). If the results support this hypothesis, our work has the potential to expand TOR-KI clinicaltrials from oncology into autoimmune diseases including MS.

Kim Green, PhD »

Treating Traumatic Brain Injuries via a novel method of brain microglia elimination


Traumatic brain injuries (TBI) are common in the very young, adolescents and the elderly, as well as in military veterans.  Patient outcome, following TBI, is variable and can affect many cognitive and motor domains leading to a loss of quality of life for those individuals.  Recent studies have shown that elevated microglia activation can be seen for up to 17 years after TBI has occurred (Ramlackhansingh et al., 2011), suggesting a long lasting degenerating process following the initial insult.  As such, it may be possible to change the course of the disease by removing these microglia.  We have recently discovered that orally administrable inhibitors of the colony stimulating factor 1 (CSF1) receptor cause rapid elimination of the vast majority of microglia from the brain, within 7 days, with no ill effects in control mice.  Furthermore, we have shown that removal of microglia facilitates cognitive recovery following a genetic hippocampal lesion in transgenic mice, suggesting that their presence hinders brain recovery.  CSF1R inhibitors have been developed for the treatment of various cancers derived from bone marrow, and are currently being tested in the clinic, and hence can be translated into other indications such as TBI.  This approach is highly novel, and far more potent that any anti-inflammatory strategy developed to date.

Specific Aims:

1) To determine if elimination of microglia via CSF1R antagonists can facilitate recovery following TBI.

We will use induce TBI in rodents and assess the effects of microglia elimination on learning, memory, and motor function.  We will use the weight drop model due to its ability to mimic a blast and produce both a focal injury at the site of the impact and a rebound injury, which occurs from the brains impact on the opposite site to the original site of impact, causing a hematoma.  Microglia will be eliminated immediately after the insult, via oral administration of CSF1R antagonists, and cognition and motor skills tested 3 months later.  We will then examine infarct size, microglia numbers and localization and synaptic markers to understand any changes that may underlie recovery in the absence of microglia.

2) To discover if there is a therapeutic window following TBI in which elimination of microglia is beneficial.

In addition, to understand the therapeutic window, we will also include groups in which we will eliminate microglia at 1 or 4 months after TBI, and then test cognition 3 months later. 

The goal of this pilot proposal is to show that removal of microglia is beneficial for recovery following TBI and to understand if a therapeutic window exists with this approach.  These data will be used to apply for larger grants from the NIH and DOD, which we hope can eventually lead to clinical trials.

Jung-Ah Lee, PhD »

A Technology Driven Safety Intervention for Older Adults New To Anticoagulation Therapy


Older adults are at substantial risk for cardiovascular disorders (e.g. atrial fibrillation, venous thromboembolism, heart failure, etc.) that require long-term oral anticoagulation treatment (OAT). However, the need for frequent visits to healthcare clinics to monitor international normalized ratio (INR), lifestyle limitations (e.g. diet and activity restrictions), and side effects (e.g. bleeding) associated with OAT has resulted to dissatisfaction and poor quality of life (QOL) and consequently poor adherence and treatment efficacy of OAT, prompting the need for alternative strategies to ensure that older adults on OAT are more optimally and consistently treated. The use of virtual reality and interactive gaming (VR-IG) - defined as “any form of human computer interface that allows the user to ‘interact’ with and become ‘immersed’ in a computer generated environment in a naturalistic fashion” – has gained recognition as an approach to deliver health education and services to individuals of all ages. Numerous studies support the effectiveness VR-IG in improving knowledge about health and awareness of risk factors, increasing self management, and reducing healthcare resource use. However, there is a paucity of research examining the effectiveness of VR-IG on knowledge, attitudes, self care management, adherence, and QOL in older adults with chronic illness; there are no studies examining the feasibility and effectiveness of VR-IG in older adults requiring long-term OAT. The current study proposes to design a theory-based, elderly-centered VR-IG intervention and test its feasibility and effectiveness using a two-phased approach. Phase 1 aims to develop a VR-IG platform using the principles of Participatory Action Research where patients provide feedback on specific program components (e.g. look and feel, game play elements, art and animation, sound track, mechanism for healthcare provider feedback, and other production features) and make decisions that will impact the design of the VR-IG intervention. In Phase 2 we propose to conduct a pilot study in 10-15 older adults (≥ 60) on long-term OAT to test the feasibility and acceptability of the study procedures and data collection instruments and assess effectiveness of the VR-IG program on several biobehavioral outcomes in preparation for a randomized clinical trial and submission of a larger extramural grant. The specific aims for Phase 2 include: 1) assess changes in cognitive function (knowledge, attitudes), emotional well-being (QOL, depression, anxiety); and perceived control; 2) evaluate changes in adherence (e.g. self-management), adverse outcomes (e.g. bleeding, thromboembolic events), and treatment efficacy; and 3) obtain data on receipt of health services and total intervention costs that can be used for future cost-effectiveness analysis. Data will be collected at baseline, three months, and six months. An extramural grant proposal to conduct a randomized controlled trial and examine the effectiveness of the VR-IG intervention vs. usual care will be submitted to the National Institute of Health-National Institute of Aging using preliminary data from this pilot study. This study will address the tremendous gaps in literature related to use of VR-IG in older adults on OAT and will potentially provide researchers and clinicians with a better understanding of the biobehavioral underpinnings associated with use of VR-IG in older adults with other chronic conditions. This information will provide evidence for rational recommendations for healthcare delivery models and strategies that can be integrated in OAT management and treatment guidelines.

Hannah Park, PhD »

Quantitative Analysis of Potential Plasma Methylation Markers for Breast Cancer


The proposed project will validate potential biomarkers for breast cancer in blood which can differentiate betweena malignant and benign breast lesion. DNA promoter (CpG) hypermethylation has been detected in the blood ofpatients with early-stage breast cancer and even before clinical diagnosis. However, the data in previous studieswere done in small cohorts and show inconsistent results, owing to variations in methodology between studies andpotential for false-positives by the methods used in most studies. Bisulfite pyrosequencing is considered the newgold standard for methylation analysis, providing high-resolution quantitative analysis (methylation data for eachCpG site) which is reproducible and easily standardized, making it an ideal method for biomarker validation.Research planA thorough review of the literature reveals six genes that are methylated with up to 100% specificity and relativelyhigh sensitivities in breast cancer, RASSF1A, DAPK1, p16, cystatin-6, BIN1, and BRCA1. These genes have beenreported to exhibit sensitivities ranging from 50-65% in serum/plasma from women with breast cancer. For thisproject, DNA methylation will be analyzed in archived plasma from women whose blood samples were obtained atthe time of biopsy for a suspicious mammogram. While some of the biopsies were found to be cancerous, otherswere found to be benign. A subset of women with biopsies that were originally diagnosed as benign havesubsequently been diagnosed with breast cancer. In addition, some of the women with breast cancer (alreadypresent at baseline or diagnosed during >14 years of follow-up) have died. The six genes will be analyzed bybisulfite pyrosequencing independently and as a panel, which will likely increase the sensitivity of the test abovethose exhibited by any one gene. Specificity is expected to remain at or around 100%.

Specific aims

We hypothesize that bisulfite pyrosequencing of the optimized 6-gene panel can differentiate between malignantand benign breast lesions and can potentially serve as biomarkers for early detection, diagnosis, and prognosis. Totest this hypothesis, we propose to study the following specific aims:1) Analyze methylation of the six-gene panel in plasma from women with malignant (n=117) versus benign breastbiopsies (n=289) by bisulfite pyrosequencing;2) Determine the most relevant genes, CpG sites, and thresholds for each gene/CpG site for which, alone or incombination, methylation status will yield the highest sensitivity and specificity for differentiating betweenpatients who were diagnosed with breast cancer at the time of original biopsy (n=117) and patients whose biopsiesdid not indicate malignancy and remained cancer-free for up to 15 years (n=254).3) Determine the most relevant genes, CpG sites, thresholds, and combinations which are associated with futurebreast cancer diagnosis (n=35).4) Determine the most relevant genes, CpG sites, thresholds, and combinations which are associated with survivalin women diagnosed with breast cancer (n=152).The proposed studies will validate or invalidate in quantitative, reproducible, and standardized terms, the six genesas potential biomarkers for early detection, diagnosis, and prognosis prediction of breast cancer.

Leslie Thompson, PhD »

ZFN-Mediated Knockdown of Mutant Htt in Patient-Derived iPS Cells

Specific Aims:

Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative diseasethat inevitably leads to death. The disease is caused by an expanded polyglutamine repeat within theHuntingtin (Htt) protein. Symptoms of the disease include a movement disorder, cognitive dysfunction andpsychiatric manifestations. Progressive loss of medium spiny neurons in the striatum and shrinkage of thecortex is observed. Current treatments are palliative and do not modify disease progression, therefore acompletely unmet medical need exists. One potential approach to treating diseases such as HD is the use ofstem cell transplantation. Our preliminary data and other studies suggest that neural stem cells transplantedinto a mouse model of HD delays disease progression. But a major drawback to this approach is that patientswould likely need to be immunosuppressed for the remainder of their lives and such treatment could itselfaffect disease progression. A potential solution to this problem would be to use patient-derived inducedpluripotent stem cells (iPSCs). Such cells, or specialized cells derived from them, could be used to treatpatients in an autologous manner without immune rejection. Since iPSCs derived from patients, althoughhistocompatible, carry a mutant allele, specialized cells derived from them may not restore function either inthe short or long term. Here we propose to engineer HD patient-derived iPSCs using the Zinc-finger nuclease(ZFN) technology to inhibit the expression of mutant Htt. Such an approach could also be applicable to manydiseases caused by mutations that are autosomal dominant.

Specifically we propose to:

Aim 1: Carry out knockdown of the Htt encoding mRNA using an short hairpin RNA (shRNA) insertedinto a safe harbor locus in HD iPSCs. Using methods for introducing genetic elements into a safe harborlocus using ZFNs, we will introduce an HD miRNA to knockdown the Htt encoding mRNA. Previous studieshave shown that a 50-65% reduction in Htt encoding mRNA and or protein ameliorates disease in HD mousemodels. The hypothesis to be tested is that generalized knockdown of Htt encoding mRNA in patient-derivedcells would “normalize” otherwise defective cells.

Aim 2: Carry out allele specific shRNA inserted into a safe harbor locus in HD iPSCs. For theseexperiments, we will introduce an allele-specific miRNA that targets the mutant HD allele into a safe harborlocus to specifically target the mutant HD mRNA. The hypothesis to be tested is that allele-specific miRNAscould specifically reduce mutant Htt encoding transcripts in patient-derived cells.

Research Plan:

We propose to reduce expression of mutant Htt protein in iPS cells derived from HD patients.With this technique, fibroblasts or other cells can be “reprogrammed” to become pluripotent cells that can bere-differentiated into neural cells. While there is recent discussion as to the extent of somatic coding mutationsand aberrant epigenomic hotspots, there is great potential in using these cells for gene therapy. As nonintegratingtechniques and other approaches to reprogramming become standard, these types of issues willlikely be overcome. To date, we have generated a total of 29 iPS lines from 5 HD subjects [(representing adultonset (~40-60 repeats) and juvenile onset (above 60 repeats)] and 2 control subjects using standard retroviralmethods. Characterization of these lines indicates that full reprogramming to a pluripotent state was achieved.Targeted integration of shRNA expression cassettes into the AAVS1 safe harbor locus in hESCs will be usedfor the integration of the Htt shRNA expression cassette into the AAVS1 locus in two patient-derived iPSCs(HDF1,HDF2) that carry a juvenile onset range CAG-repeat. Both published shRNA sequences and thecorresponding miRNA developed by the Davidson lab (collaborator) will be subcloned into the AAVS1 donorconstruct and co-transfected with AAVS1 ZFNS into iPSC lines. Both miRNA and shRNA will be tested in iPSlines to determine the degree of expression required. As a proof of principle and to both assess efficiency andfor enriching (via cell sorting) the cells that have the correct integration, a promoterless GFP coding sequencewill be included in the donor construct sequence. Knockdown will be evaluated by qPCR. For ultimate clinicalapplication, the GFP sequence will not be used in the donor constructs and screening will be accomplished bymonitoring integration by Southern blotting and expression by qPCR. By using GFP in initial studies, we willdetermine the efficiency of transfection and can estimate the number of clones required to select integrants.For the allele-specific knockdown approach, we will collaborate with Dr. Neil Aronin to incorporate a mutantallele specific shRNA or miRNA cassette into the AAVS1 donor construct using approaches as in Aim2.