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RS1-00212-1: Targeting lentiviral vectors to modified hES derived dendritic cells

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

We hypothesize that human embryonic stem cells represent a potentially scalable source of human dendritic cells that could be used to treat a wide array of diseases including HIV/AIDS and cancer. In fact dendritic cell based therapies have shown some promise in early trials, but they are largely limited by the numbers of cells available for treatment protocols. We propose here to develop a potentially unlimited source of human dendritic cells from human embryonic stem cells. More importantly, we propose to modify these cells to express a novel cell surface molecule that can be used for targeted delivery of specific proteins. Ultimately we envision using these cells to direct primary immune responses. The studies described in this SEED proposal are directed towards HIV specific immune responses, but would be widely applicable to other pathogen-based diseases, as well to tumor associated antigens.

Statement of Benefit to California (provided by applicant)

Dendritic cell immunization, dendritic cell-based gene therapies and immunotherapies are being explored as treatments for a number of diverse human diseases, particularly in settings that are refractory to conventional therapies. Unfortunately, these protocols are directly limited by the ability to generate sufficient quantities of dendritic cells ex vivo. Human embryonic stem cells represent a potentially scalable source of dendritic cells that could be used in these settings. For instance, they could be used for the prevention and treatment of pathogen based diseases such as HIV/AIDS, Hepatitis C and Influenza. In addition, development of a source of self renewing dendritic cells could dramatically advance patient specific protocols for the treatment of cancer. Taken together these diseases and their treatments impact Californians personally and economically. The development of improved treatment and prevention modalities by harnessing the potential of human embryonic stem cells would represent a major benefit to the lives of all Californians.

Review

SYNOPSIS: The investigator proposed to use gene transfer methodology to genetically engineer dendritic cells derived from hESC to enhance their transducibiliy with targeted lentiviral vectors. The gene for a modified factor will be introduced and gene transfer will be initially evaluated using a GFP marker. If successful the system will be utilized to introduce portions of the HIV genome designed to express specific antigens.

SIGNIFICANCE AND INNOVATION: This work is innovative in that it utilizes state-of-the-art methodology in an effort to derive antigen-loaded dentritric cells from hESC-derived dendritic cells to target possible immunotherapeutic proteins or peptides via targeted lentiviral vectors for immonological stimulation. Innovation is dimished because the investigator fails to adequately justify the rather complex experimental strategy. The work is potentially signficant (but modest) because of its focus on enhancing the immune response to HIV and tumor antigens, and because there may be other sources of dendritic cells, and other means to target immunogenic peptides for therapies. It may not produce results of generalizable significance.

STRENGTHS: Overall, this is a well-conceived proposal. The three specific aims are all logical and coherent and fit the scope of this type of SEED grant proposal. The ability to produce functional dendritic cells from human ES cells could be quite feasible and this proposal is assisted by strong collaborations, including one investigator who has significant experience in hematopoietic cell differentiation of human ES cells. The Principal Investigator (PI) also has sa trong background in HIV and possible novel immunotherapies against HIV, and experience with the relevant methodology. There is a well-delineaated expermental approach, and the aims of this proposal, if completed, would provide important results for subsequent studies and potential funding.

WEAKNESSES: A reviewer is not convinced that the proposal’s sequential tranduction strategy is necessary to achieve the stated goals. An alternative approach would be to introduce vectors encoding the antigens of interest and then differentiate hESCs into dendritic cells by the proposed differentiation scheme.

Little discription is provided of the strategy to obtain expression of the key antigenic epitopes. What part of the HIV genome will be included? How will the expression cassettes be designed to insure high level production of the processed antigenic peptide?

In addition, there are a few technical weaknesses. In Specific Aim 1, PI proposes to express a protein in hESC using either electroporation or lipid-mediated transfection. However, this is technically difficult to do with hESC as the efficiency of these methods and silencing of transgenes expressed in such a manner can be significant problems. Most studies for stable transfection of hESC use lentiviral-based vectors. If that would need to be done here, it is unclear whether that would interfere with subsequent studies to use targeted lentiviral vectors in Specific Aims 2 and 3.

Another weakness is in Specific Aim 3 where they will be testing antigen-specific immune responses. While this seems feasible based on other methods, it is a little unclear whether the lentivirus itself will also contribute peptides that could interfere with the subsequent analyses.

Overall, a reviewer believes this proposal to be potentially significant. There is a concern that the PI may be ignoring other possible strategies that could strengthen these studies; this is problematic. It is unclear why either dendritic cells from peripheral blood or derived from other haematopoietic stem cells such as umbilical cord blood would not be as good or potentially better than use of hESC-derived dendritic cells. It is also unclear if this would develop into the anticipated conclusion to produce hESC dendritic cells that can be targeted for immunotherapy. Whether or not this would actually be a viable therapy is in question.

This proposal would benefit from an acknowledgement of alternative methods of producing these types of targeted immune responses with an emphasis on why the use of hESC-derived dendritic cells is essential for this type of work. There should also be consideration of other means to generate stable transfectants from hESC or evidence that these transfected cells lines have been generated.

DISCUSSION: One reviewer stated that the design and concept of the proposed study is quite novel and interesting. There was a question as to why the applicant proposed to derive dendritic cells from hESC and then transfect them as opposed to transfecting hESC and then deriving dendritic cells. Also the question arose as to why use the applicant would use dendritic cells derived from hESC rather than from other sources. Discussion highlighted some points of the proposal, and the methodology was regarded as feasible but a discussant offered that other strategies would be likely to work as well.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Lansing, Sherry