The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of important work is describing the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we will examine how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We will characterize the programs of regulation controlled by these proteins. In particular, we will focus on those parts of the PTB regulated splicing program that affect cell proliferation and the ability of ES and NP cells to self renew. From this work, we will advance our understanding of how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.
Neurological diseases affect millions of patients in California and elsewhere. For example, spinal muscular atrophy (SMA) is one of the most common genetic causes of infant death and has no effective treatment. SMA and other neurological diseases are caused by errors in the cellular process of pre-mRNA splicing. One promising strategy for neurological treatments is in cell replacement therapies using hES and iPS cells as source material for regenerating normal neurons in place of those lost to the disease. Another therapeutic strategy for SMA is in drugs that alter the splicing process to improve its efficiency in diseased cells. This project will examine the splicing process in normal hES cells and how it is regulated when these cells differentiate into neuronal progenitor cells and neurons. This will provide essential information on the biology of stem cells needed to move towards various therapeutic applications. The project will also provide a system for drug discovery in the new field of splicing targeted therapeutics. This work will help California to continue to lead in these areas of basic research, as well as provide the state with a head start in biotechnology and pharmaceutical development for the practical application of these discoveries.
This proposal focuses on alternative splicing, a regulatory step in the production of protein from RNA, and its role in human embryonic stem cell (hESC) differentiation into neurons. Alternative splicing affects the expression of the majority of human genes and is particularly prevalent in the nervous system. The applicant proposes to comprehensively profile alternative splicing in hESCs as they differentiate into neural progenitor cells (NPCs) and neurons, while focusing on a splicing regulator, polypyrimidine tract binding protein (PTB), and its neuronal homolog, nPTB. In Aim 1, the applicant will use novel technologies to profile genome-wide alternative splicing in hESCs, NPCs and differentiated neurons and map the genome-wide RNA binding of PTB and nPTB. In Aim 2, the applicant proposes to identify specific RNA targets of PTB and nPTB and confirm their regulation by these proteins. Finally, in Aim 3, the applicant proposes to characterize the function of these identified RNA targets in hESC and NPC proliferation and differentiation.
The reviewers agreed that this proposal addresses an interesting and important question in stem cell biology. They noted that the experiments are mostly descriptive in nature and so the proposal’s significance is difficult to assess, as it will depend on the results obtained. Nevertheless, reviewers were confident that the project would generate large amounts of high-quality data that could have a major impact on the field of regenerative medicine. They found aspects of the proposal to be innovative, particularly the use of novel technologies and the focus on PTB and nPTB. However, reviewers noted that at least two papers have examined alternative splicing in hESCs, including an important study published recently by Salomonis, et al. which was not cited by the applicant. One reviewer felt that the proposal would be more innovative if it examined hESC differentiation into different neuronal subtypes and analyzed the roles of PTB and nPTB in these later cell fate decisions.
Reviewers found the research plan to be well-considered and feasible. They appreciated the use of cutting-edge sequencing technologies and felt the applicant makes a good case for their advantages over hybridization-based array technologies. However, reviewers found the preliminary data sparse and couldn’t determine whether they described experiments in cells of mouse or human origin. They also raised a few concerns about the experimental design. They noted that progress in Aim 2 will be highly dependent on Aim 1 and alternative approaches are not discussed. With regard to Aim 2, reviewers cautioned that, given previously published data in mouse ESCs, knockdown of PTB may have profound effects on cell viability and/or differentiation, making it difficult to use this technique to verify PTB-regulated exons. A final concern with any such ambitious, genome-wide approach is the difficulty of data prioritization and validation of targets. However, reviewers noted that the applicant addresses this issue and they were satisfied with the proposed strategy.
The reviewers described the PI as an internationally recognized expert in the field of alternative splicing with an outstanding publication record. They did note that the relative lack of hESC expertise on the research team could be a challenge. A number of collaborators are named in the application but there are no accompanying letters of support. Still, reviewers were confident that the PI and research team have the resources and expertise to complete the proposed studies.
Overall, reviewers felt that this proposal is likely to produce important new information about hESC biology and neuronal differentiation. They raised some minor concerns about the experimental design but were confident in the ability of the outstanding PI and research team to generate large amounts of high-quality data that will benefit the field.
- Ali Brivanlou