Tools and Technologies I
The ability to transfer therapeutic genes into human embryonic stem cells (hESC) has tremendous value for the permanent correction of genetic diseases. Gene-corrected hESC have the potential to be used for the treatment of inherited and acquired diseases. Adeno-associated virus (AAV) is human virus that does not cause any known disease. We and others have used AAV as a tool to deliver therapeutic genes to cells by replacing the viral genes with therapeutic genes. Only the virus backbone and shell is required for it to function as a gene delivery tool. Genes that are delivered by AAV can become part of the cells genome or remain as unincorporated DNA in the nucleus. Regardless, it is clear from collective experience over four decades that AAV is safe and does not harm the host. We recently found a new family of AAV viruses in human blood cells. We hypothesize that this family of AAV will make good vectors for the transfer of genes to hESC. We have also recently obtained a panel of next generation AAV vectors recently derived at the University of Florida that were shown to be highly efficient at gene transfer at lower doses with no associated toxicity. Here we propose to test these two families of novel AAV vectors for their ability to transfer genes to hESC. We shall compare members of these two AAV families to determine which is best for long-term gene transfer to hESC. Developing these gene transfer tools for therapeutic use requires making certain that the normal properties of hESC are not altered, including their primitive pluripotent status and their ability to differentiate normally into other cells. Moreover for treatment of specific diseases we shall evaluate members of these AAV families to transfer genes to progenitor cells of specific types derived from hESC. These will include cells that have the capacity to make nerve and muscle cells. It is also important to determine whether the genes transferred to the cells become a part of the cells genome or may be lost over time. We propose experiments designed to address this. Based upon our previous work with blood forming stem cells, we fully expect to identify one or more members of these AAV families that are able to mediate permanent genetic correction of hESC. We will also confirm that such gene-modification is safe. If some members of these families of AAV do not cause the transferred genes to become a permanent part of the cells’ genome, they will be identified as ideal candidates for future work on the transient gene transfer. When completed, this research will identify tools to introduce genes into hESC in a safe and effective manner.
Statement of Benefit to California:
This research develops genetic therapeutic tools for human embryonic stem cells (hESCs). We propose to develop novel gene transfer tools that can be used safely and efficiently in basic and clinical research employing hESCs. It is expected that tools developed from this research will eventually be used for treatment of a variety of genetic diseases that are currently incurable. Diseases addressable with these tools include a number of genetic diseases that are inherited or acquired through mutations which affect Californians, many of which exact a heavy social and economic toll. The tools that will come out of this proposal do not focus upon one specific disease; rather we propose to develop safe and efficient gene transfer tools for hESCs that could then be used for many conditions with an identifiable genetic basis. Moreover these new tools could also be used to study the processes involved in early human development, which in turn would provide insights into both normal and abnormal growth processes. Upon completion of this research, we expect to have a panel of safe and effective tools for the genetic modification of hESCs that will be available to researchers in the state. Thus the work proposed here should have an overall impact on public health in California in terms of advancing both therapeutic approaches and basic science of genetic medicine through the use of hESCs.
This proposal aims to evaluate adeno-associated virus (AAV) as a means of genetic modification of human embryonic stem cells (hESCs). The applicant proposes to test a number of novel AAV vectors derived from human hematopoietic stem cells (HSCs), with and without tyrosine modification, for the ability to sustain transgene expression in hESCs without toxicity. The applicant notes that AAV genomes sometimes persist in episomal form following transduction, rather than integrating into the host genome. As vectors that promote episomal expression are not suitable for long-term hESC transduction to make stable lines, the applicant will also evaluate the potential for the vectors as tools to reprogram differentiated cells into induced pluripotent stem cells. The reviewers agreed that the proposal’s primary goal of identifying novel methods for safely introducing transgenes into hESCs is an important one. However, they were not convinced by the rationale for the approach and expressed doubts about the project’s likelihood of success. The reviewers praised the research team and commented that the applicant and main collaborator are well qualified to carry out the work. The reviewers agreed that this proposal would have high impact in the field if successful. Safe methods for introducing genetic material into hESCs will be required when hESC-derived cells are developed for clinical treatment of genetic disorders. The applicant described the advantages of AAV in terms of its relative safety, but at least one reviewer would have appreciated a more thorough comparison with other classes of vectors, such as lentiviruses, as well as some discussion of potential disease targets. This reviewer commented that in situ repair via homologous recombination, combined with recombineering or zinc finger nucleases to enhance efficiency, or non-integrating self-replicating vector approaches might be more promising approaches for generating clinically suitable stem cell therapies. Reviewers felt that study design and approach were mostly straightforward and well thought out but expressed concern that the research design would not lead to the intended outcomes. One reviewer found the proposal overly ambitious, given the number of AAV serotypes to be studied and the apparent lack of prioritization based on their capacity to infect hESCs. Reviewers also questioned the rationale for using AAV vectors with no proven tropism for hESCs. The premise of the proposal is that the capsid proteins of naturally-occurring AAV serotypes isolated from human HSCs and tyrosine-modified AAV will have an increased capacity to infect and/or stably express transgenes in hESCs compared to the current state-of-the-art AAV vectors. However, the reviewers commented that the applicant offers no compelling argument to support this premise. It is not clear that the tropism of the human AAV serotypes to be tested is any different than that of AAV2 commonly used as a gene transfer vector. The fact that the naturally-occurring human AAV serotypes proposed for use in this application were isolated from HSCs and can infect HSCs does not imply enhanced tropism for even these cells, until proven, let alone hESCs. The reviewers did not feel that the preliminary data provided sufficient rationale for the chosen approach. Reviewers thought that testing tyrosine modified AAV vectors was a promising idea, but would have liked more background information. They also would have appreciated a discussion of the possible silencing of AAV transgenes, which has been previously seen in pluripotent cells that are differentiated and transfection. The reviewers characterized the research team as very strong. The applicant and primary collaborator have excellent track records in AAV vectorology and stem cell biology. The budget seems reasonable and justified. Overall, this proposal addresses an important roadblock in stem cell research but the reviewers expressed serious doubts about the feasibility of the chosen approach within the short time frame of the initiative.