Funding opportunities

Directed Stem Cell Recruitment into the CNS in Response to Coxsackievirus Infection

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$1 788 000
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Improved and more effective treatment strategies to alleviate the clinical symptoms associated with multiple sclerosis (MS) remain problematic. A better understanding of stem cell migration across the blood-cerebral spinal fluid (CSF)-barrier and targeted differentiation may assist clinicians and researchers in maximizing the efficacy of stem cell based therapies. Many previous studies utilizing stem cell therapy for tissue regeneration have relied on the seemingly inherent ability of stem cells to migrate and differentiate somewhat haphazardly into predetermined areas in damaged tissues. However, the random inoculation and migration of stem cells, regardless of regenerative potential, is unlikely to lead to the most efficient recruitment of stem cells and subsequent repair of critically damaged regions . Utilizing our neurotropic viral infection model which appears to induce the recruitment of peripheral stem cells, we will attempt to characterize stem cell migration across the blood-CSF-barrier and determine necessary recruitment factors upregulated during infection. Additionally, we will test the ability and advantages of utilizing an attenuated recombinant coxsackievirus as a novel viral vector for gene transfer into stem cells. The ability to insert foreign genes into stem cells may assist in proper stem cell differentiation and protection from the host immune response. Finally, we will evaluate the therapeutic use of stem cells transduced with an attenuated recombinant coxsackievirus utilizing a relapsing-remitting mouse model of MS. MS is thought to be an autoimmune disease in which the loss of myelin contributes to central nervous system (CNS) damage and clinical disease. Stem cell transplantation may be a therapeutic approach for the re-establishment of myelinated axonal fibers and proper neuronal function. The addition of foreign genes into stem cells may help protect the newly differentiated cells from suffering from the same fate as their non-functioning neighbor cells. We hope that the proposed research will assist in the development of novel reagents for optimized stem cell therapy in patients suffering from neurological diseases.
Statement of Benefit to California: 
Approximately 400,000 Americans suffer from the neurological symptoms associated with multiple sclerosis (MS). The therapeutic use of stem cells may provide the basis for improving the lives of patients suffering from MS, and a variety of other neurological diseases. However, much remains to be understood regarding productive stem cell migration and proper differentiation of oligodendrocytes in the host. Oligodendrocytes are essential cells in the central nervous system (CNS) that help to protect the axons of nerve fibers by producing a fatty tissue called myelin. In MS, myelin is lost in many region of the CNS thereby producing sclerotic lesions. Hence, the ability to promote new myelin formation in damage regions by the addition of new oligodendrocytes may be a potential therapy for the alleviated the effects of clinical disease. The project will provide new insights on how stem cells may traverse through the blood-brain-barrier and differentiate into the various cell types found within the CNS. Furthermore, we will test the ability of using a well characterized coxsackievirus that efficiently infects stem cells as a viral gene delivery system. Our hope is that the information gained from our efforts will point to new directions, whereby optimized stem cell therapy may be of great benefit to those suffering from MS, and other neurological disabilities.
Review Summary: 
SYNOPSIS: This is an application for support to follow up studies conducted by the PI showing that the picorna virus, coxsackievirus B3 (CVB3), preferentially infects proliferating neural stem cells in the neonatal CNS and recruits nestin-positive myeloid cells through the blood-cerebral spinal fluid barrier; and that CVB3 RNA persists in the CNS for months after the initial infection. The recruited cells leave the choroid plexus, migrate across the ventricles, enter the ependymal cell layer, exhibit characteristics of type B neural stem cells and serve as highly susceptible hosts for CVB3 infection. The applicant plans to use this neonatal virus infection model as means to explore chemoattractant molecules that influence the migration of bone marrow derived cells into the CNS, and to utilize a recombinant CVB3 virus as a viral vector as a means of transducing stem cells. The applicant will then utilize the virus to transduce neural stem cells which will be used to attempt to ameliorate EAE, a mouse model of multiple sclerosis. Three specific aims are proposed: Aim 1 will determine cell populations and chemoattractants responsible for stem cell recruitment into the CNS following CVB3 infection. Aim 2 will evaluate an attenuated or defective recombinant rCVB3 as a viral expression vector. Aim 3 will determine the ability of CVB3-transduced stem cells to diminish disease in a mouse model of MS. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is an interesting application from a young investigator with insight into the potential importance of findings somewhat tangential to coxsackie meningitis. This work originates from the PI’s studies in Whitton’s lab and his interesting paper in J. Neurosci (2005), where he showed that the rCVB4 virus targets proliferating neural progenitor cells in the neonatal CNS.The PI provides preliminary data showing that CVB3 leads to the recruitment of stem cells from the periphery that join the neural stem cell population located in neurogenic regions of the developing brain. As the PI notes, the ability of bone marrow-derived cells to contribute to repair remains controversial because of issues related to whether these cells actually transdifferentiate or are involved in cell fusion. There are a number of strengths to this proposal. The Preliminary Data of the PI is of interest and involves creative experiments. The identification of the relationship between a virus infection and stem cells is of interest and provocative. The specific aims of this present proposal, however, seem somewhat disconnected. Although all three specific aims involve CVB3, they do not fit together in other ways: specific aim 1 uses CVB3 to identify chemoattractants attracting myeloid cells that become nestin+; specific aim 2 focuses on the use of CVB3 for gene delivery into SCs; specific aim 3 examines the effect of varied factors on EAE. It is a major concern that the PI states right at the beginning of the proposal that the experiments may be risky The first specific aim involves a controversial issue in SC biology. The relationship between myeloid-derived nestin+ cells and SCs remains unclear. His evidence for trandifferentiation of myeloid cells into nestin+ cells is hard to conclude from the “postage stamp” size figures in the preliminary results section. It is not entirely clear how he would use the information generated form Specific Aim 1 in therapeutic approaches. Will the chemoattractants that he finds unregulated in the neonatal mice have relevance to the recruitment of stem cells in the adult CNS? The use in specific aim 2 of CVB as a vector for gene delivery may have problems related to maintenance and stability of the foreign gene. The reviewers were uncertain why the PI chose CVB for these experiments. The proposal generally did not seem well-unified. Beyond these general critiques, detailed concerns with the experimental approach are discussed below. In specific aim 1a, the PI will identify the chemoattractants involved in SC recruitment into the CNS following CVB3 infection. The PI notes that this information will be important in facilitating SC delivery from the periphery. In order to carry out this aim, the PI will use RNase protection assays to measure levels of chemoattractants. Cytokines and type 1 Ifn response genes will be anyalyzed by Dr. Iain Campbell in Sydney, Australia. The PI also writes about using an Affy geneChip with >47,000 transcripts. It was unclear in what experiments the PI would use this chip. The upregulation of factors will be confirmed with real time RT-PCR; in addition, immunohistochemistry will be performed to determine the cell that is producing the molecule of interest. The PI will also use mouse mutant models to clarify whether independent upregulation of specific genes are key in attracting stem cells into the CNS. For example, he will determine whether nestin+ myeloid cells can be mobilized independent of CVB3 in a transgenic mouse that expresses IP10 under the GFAP promoter - and an IP10 knockout mouse will be used to determine the effect on nestin+ myeloid cells following CVB3 infection. There are a number of concerns related to the latter experiments (and ones involving other chemoattractants). Modulation of IP10 (or other immune-related factors) may have a primary effect on the CVB3 infection which has a secondary effect on many chemokines and cytokines, complicating the interpretation of the data. In addition, these kinds of experiments may not provide clearly positive information if more than one factor may be necessary for the transformation of the myeloid cells. In the second part of specific aim 1, the PI will identify the bone marrow cell populations involved in SC recruitment into the CNS following CVB3 infection. The PI will intra-hepatically inject (from adult H2K-zFP mice, which express GFP under the control of the H2K promoter element) purified populations of hematopoietic SCs, mesenchymal SCs, and bone marrow cells depleted of the latter two populations; recipient 1-day-old mice will be infected with a CVB3 that expresses RFP. Brain and liver of the recipient mice will be evaluated at days 1, 2 and 6 by fluorescence microscopy in order to quantitate nestin+ cells. In addition the PI will inject donor liver cells, a major hematopoietic organ early in life, from newborn H2K-zFP mice. Preliminary data are provided; however, there appear to be very few cells that entered within the CNS and none are infected with virus (and no nestin staining is shown). It remains unclear whether a sufficient number of injected cells will enter into the CNS, complicating the successful completion of these studies. A small number of cells will also complicate the PI's plan to derive neurospheres from the nestin+ myeloid cells. In specific aim 2, the PI will evaluate an attenuated or replication-defective CVB3 vector as a vector for gene delivery into SCs for experiments planned in specific aim 3. There are several questions and issues that arise with respect to this specific aim. The rationale for choosing CVB3 provided by the PI includes the stability of the genome and its long-term expression and persistence. However, there are reports of instability of foreign genes placed into picornaviruses, including enteroviruses as well as cardioviruses - so that part or all of inserted genes may be lost, especially during proliferation of SCs. There is limited space within the enterovirus genome, so that large inserts may foster deletion or limit infectivity. Furthermore, it is not clear how common persistence of enteroviruses is – or whether this is an advantage or disadvantage to pexrsistence. It was unclear why the PI chose CVB3 for gene delivery since there are other viruses that are frequently used as vectors. Would another virus be preferable to use, especially one that is more commonly used? In order to make the attenuated CVB3, the PI will replace the 5’UTR of the infectious clone with a 5’UTR of an attenuated CVB3. The attenuated virus needs to infect cells (e.g., NSCs in specific aim 3), but not kill the cells. Considering the propensity of CV to mutate, won't there be issues and concerns about mutations occurring in the attenuated virus potentially making it more virulent? These issues raise concerns about whether this virus would be made sufficiently attenuated in order to be an attractive vector for gene delivery into SCs that are then injected into humans. The reviewers thought that the PI should have dealt with these issues in the proposal. In a second approach, the PI will generate a replication-incompetent CVB3 vector that has replacement of P1 with a foreign gene; replication occurs following transfection of the defective genome into P1-expressing HeLa cells. This seems like a better approach to the one involving an attenuated virus, although there still may be instability issues with respect to maintenance of the inserted genes, as noted above. Also, one wonders whether there will be toxicity issues related to P1 expression in HeLa cells. Again, why choose CV since there are other replication-deficient viruses in common use? In specific aim 3, the PI will use the attenuated CVB3 or defective CVB3 vector to determine whether CVB3-transduced eGFP+ NSCs have an effect on EAE. An issue with respect to this Specific Aim was its relationship with the specific aim 1. What is the relationship between a treatment of EAE and the identification of chemokines that guide myeloid-derived nestin+ cells? In specific aim 3a, the PI will insert olig2 into the attenuated virus or defective virus vector in order to determine the effect on the number of oligodendrocytes in EAE. In specific aim 3b, the PI will test the effect on EAE of eGFP+ NSCs that are transduced by attenuated CVB3 or a defective CVB3 vector that contains transcription factors or immune evasion genes. The immune system’s effect in EAE is complex and involves multiple factors. How will the PI decide which foreign genes to insert? QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is ideally suited to carry out this project. His time table and enthusiasm for this project indicate five years of funding would successfully recruit a virologist into a stem cell scientist with specialized tools for stem cell fate determination. Dr. Feuer just recently assumed his present position as Assistant Professor in the Department of Biology at San Diego State University. The PI received his PhD in 1999 from the University of Nevada and then pursued a post-doctoral fellow in J. Lindsay Whitton’s lab at Scripps from 1999-2004; he was Senior Research Associate in the same lab from 2004-2007. The Whitton lab is an outstanding one with respect to training in virology and immunology. He has just been appointed an Assistant Professor at San Diego State University The PI was first or last author on ~5 refereed papers (in American J. Path., J. Virology, J. Neuroscience) since 2002; these papers have focused on coxsackievirus pathogenesis studies and are of high quality. One wonders whether the publication record is relatively modest considering the fact that he is 8 years out from his PhD. He was recently awarded an RO1 grant related to coxsackievirus latency, immune activation, and pathogenesis in the CNS. He was named a Young Investigator Award Finalist at EUROPIC 2005 – 13th Meeting of the European Study Group on the Molecular Biology of Picornaviruses. The applicant was trained principally in neurovirology, and with his finding of tropism of the CVB3 virus for neural progenitors, now wants to turn his attention to stem cells and the use that this virus might have in their infection and transduction in disease. In the first instance he targets MS, but he hopes that the results he generates may apply to neurodegenerative disorders, such as Parkinson’s disease in the future. Of note is that he is already succeeded in landing an R01 working in a similar area.It is not clear whether the PI had any formal training in the SC field which may be both a plus or negative with respect to this proposal, i.e., CIRM has the opportunity to strengthen an interest in SCs in someone who has expertise in immunology and virus pathogenesis studies. There was some concern that Dr. Feuer would be somewhat isolated with respect to his scientific environment. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. Feuer recently assumed his present position as Assistant Professor in the Department of Biology at San Diego State University. He is presently in a recently renovated lab of ~1000 sq. ft. and received a start-up package of $280,000. There is a FACS facility, Microchemical Core facility, a vivarium including a BSL-2 facility, and access to a confocal microscope. Dr. Feuer holds an Adjunct Assistant Professor status at the Scripps Research Institute which gives him access to these core facilities, including a DNA array core. San Diego State University has the largest Biology Department compared to similar departments in the CSU system. SDSU recently opened up a BioScience Center whose mission is the relationship between infectious disease and cardiovascular disease. There is a tenure-track committee. It would be most advantageous for Dr. Feuer to have an interactive scientific environment, so that the PI has contact with individuals in the SC field on a frequent basis. Although SDSU appears to be a nurturing environment, one is concerned that Dr. Feuer specifies only two individuals in his proposal who have a general interest in SCs at this institution. One wonders whether Dr. Feuer may be somewhat isolated in his Department and SDSU. It is true that his prior collaborations are bound to be helpful, but it remains unclear how effective these will be. DISCUSSION: The reviewers were in agreement that the relationship between the Coxsackie virus and neural progenitor cells is intriguing. However, the significant concerns with the research proposal reduced enthusiasm for the application. Reviewers felt that the proposal was not well integrated, citing that specific Aim 2 doesn't belong in this proposal, and it is unclear how well intergrated Aim 3 is with the others. The main problem with the application is with the choice of coxsackie virus (CV). Other concerns with the application included: that the choice of markers may not be specific to neural cells, the PIs choice of language to report on findings in the EAE model suggest naivete with this aspect of the project, and eight years out the PI doesn't have any real home-run papers. A single case was made in favor of the application, that since SC migration into and out of the brain is an unknown, finding any chemoattractant would be a real step forward for the field. With respect to the level of institutional support, opinions among the reviewers differed. Comments ranged from one reviewer noting that institutional support and mentorship are not as strong in this proposal as in others, to another reviewer saying that he institutional support seemed quite high, and that SDSU is trying to put a program together. The discussion closed with the impression that the insufficient biology and research plan could not overcome the interest in the initial concept.

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