Human cytomegalovirus (HCMV) is the major cause of birth defects, almost all of which are neuronal in origin. Approximately 1% of newborns are infected, and of the 13% that are symptomatic at birth, 50% will have severe permanent hearing deficits, vision loss, motor impairment, and mental retardation. At least 14% of asymptomatic infants also will later show disabilities. Much of this effect is likely caused by HCMV affecting neural development in the fetus.
Embryonic stem cells are an excellent source of human progenitors, which are cells that can turn into mature neurons i.e. neural differentiation. We know from published cell culture studies that HCMV affects neural progenitor cells during neural differentiation, but it is unclear as to what are the underlying molecular mechanisms for its effect. A major goal of our research is to understand at a high-resolution how HCMV controls the way neural progenitors become proper neurons. Elucidation of the genes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns.
The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
Human cytomegalovirus (HCMV) is the major viral cause of birth defects. In 2009, there were 526,774 births in California, resulting in congenital HCMV infection in approximately 5,200 newborns, with at least 800 infants expected to have long-lasting disabilities. Congenital cytomegalovirus infection is the most common nongenetic congenital cause of deafness. In contrast, before the development of the rubella vaccine, less than 70 infants per year in the entire US were reported to have congenital rubella syndrome, also associated with deafness. The burden to families and the economic costs to society of congenital cytomegalovirus infection are immense, and there is no vaccine available. Our proposed research serves to form the basis of future therapies to ameliorate, or even reduce this medical burden.
The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals who receive transplants of organs and stem cells from other individuals. Infection in these transplant recipients often results in severe disease and rejection of the transplant. The California Institute for Regenerative Medicine has made a major commitment to provide funding to move stem cell-based therapies to clinical trials. The goal of using stem cell transplantation to treat neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
Our research will provide the knowledge base to understand the genes that are changed during HCMV infection of human neural progenitors and neurons. It will also provide a foundation for studies of how other viruses will affect human neurons, and likely, other cell-types. Intellectual property from this work will feed into opportunities for antiviral strategies and increased jobs in biotech for Californians.
The objective of this proposal is to examine the effects of infection with the human cytomegalovirus (HCMV) on the neural lineage specification and maturation of stem and progenitor cells. Congenital HCMV infection is a major cause of birth defects, likely due to effects on neural development. HCMV infection of neural progenitors has recently been shown to promote their differentiation and therefore may be risk factor for neural progenitor cell (NPC) therapies. In order to achieve their goals, the applicants have divided the project into three specific aims. In Aim 1, the applicants propose to investigate the stage of differentiation when human embryonic stem cell (hESC) derived neural precursor cells become susceptible to HCMV infection and determine the effects of HCMV on differentiation. Aim 2 calls for genomic analysis of RNA transcripts to look for differences in RNA splicing and microRNAs following HCMV infection. In Aim 3, the applicants propose to select candidate RNA transcripts identified in Aim 2 for use in gain-of-function or loss-of-function studies to examine the effects of those RNAs in defects in differentiation of HCMV-infected cells. Aim 3 will also include analysis of effects of HCMV infection on the anaphase-promoting complex (APC) on differentiation.
Significance and Innovation:
- HCMV infection represents an unmet medical need. Determining the susceptible cell types for HCMV infection and the consequences for neural differentiation are significant and of potentially high impact.
- The data set derived from the profiling methods is likely to be descriptive and lead to studies of an exploratory nature without elucidating strong mechanistic insights, thus, reducing the impact of the proposed studies.
- The application of the genomic techniques may yield novel candidate mediators and the combination of analyses of a viral infection of human progenitors was considered novel.
- The proposed research is innovative and focuses on an understudied area; however, the application does not clearly describe the rationale for much of the proposed research nor explain how the experiments will provide mechanistic insights to the problem. For example, the approaches addressing changes in splicing are certainly innovative, but the biological rationale for looking specifically at splicing in the context of how HCMV infection affects neuronal differentiation is lacking.
Feasibility and Experimental Design:
- The strong preliminary data on the molecular analysis of HCMV infected human fibroblasts support the feasibility of the proposed studies and clearly demonstrate the applicant has the tools and techniques in hand to perform the described experiments.
- The specific neuron subtypes that will be generated and subjected to HCMV are not clearly defined, and there is no data on glial differentiation or purification. Given the selectivity of the infection with some cell types particularly affected, the team should provide characterization of all these cell types.
- The applicant already has reasonable candidates and proposes backups for Aim 3 if the targets from Aim 2 do not pan out. However, the proposal does not outline a clear plan for following up on hits nor is there a clear rationale for selecting hits. This lack of experimental detail and rationale renders the proposed studies as descriptive rather than mechanistic in nature.
- The proposed experiments do not allow for the applicant to easily identify which observed changes impact HCMV disease and which do not. Reviewers suggested the use of multiple viruses to try to explain why HCMV infection causes such devastating disease or the inclusion of a non-pathogenic control to increase the likelihood that the applicant will be able to distinguish changes attributed to infection of neurons verses those specific to the pathogenic outcomes of human HCMV infection.
- Reviewers valued the approach focusing on components of the APC as it offers a more clearly defined hypothesis. They questioned however, whether hESC derived NPCs are a better population to address the question than primary NPCs.
Principal Investigator (PI) and Research Team:
- The PI is an accomplished and well-funded virologist with an excellent publication record in top journals and a long history studying HCMV.
- The research team overall was seen as very strong with the appropriate expertise, including cell differentiation and genomic analysis. The preliminary data demonstrates that effective collaborations with all of the necessary technologies are already in place.
Responsiveness to the RFA:
- The proposal is responsive to the RFA as it attempts to address basic questions and potential mechanisms related to HCMV mediated changes in neural development and differentiation.