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RS1-00172-1: Genetic modification of the human genome to resist HIV-1 infection and/or disease progression

Recommendation: Recommended if funds available
Scientific Score: 80

First Year Funds Requested: $321,326.00
Total Funds Requested: $642,652.00

Public Abstract (provided by applicant)

The proposed studies describe the genetic approaches utilizing human embryonic stem cells to suppress and/or eliminate the expression of the human protein CCR5. CCR5 is found on the surface of white blood cells. HIV-1 attaches to CCR5 and uses CCR5 to enter into its target cells. Our approach is to utilize established as well as new non-established approaches to prevent CCR5 from appearing on the surface of the cells. If CCR5 is not present, HIV-1 cannot infect the cells. Interestingly, this concept has already been proven in nature. Approximately 1% of the Caucasian population is genetically deficient for CCR5 and these individuals are resistant to HIV-1 transmission. Their white blood cells, when placed in culture, also resist HIV-1 infection in the laboratory. As such, we believe that our approach can be used to protect high risk individuals from HIV-1 infection as well as impede or stop progression of disease in those individuals already infected.

Statement of Benefit to California (provided by applicant)

According to the Centers for Disease Control, California is second only to New York of individuals living with AIDS. Developing means to stop HIV-1 infection and cure those individuals already infected with HIV-1 is of paramount importance for the state of California.

Review

SYNOPSIS: The investigator proposes to utilize gene modification protocols to engineer human embryonic stem cells (hESC) that, upon hematopoietic differentiation, generate cells that are resistant to Human Immunodeficiency Virus (HIV) infection. The applicant proposes to use methodologies and vectors already developed and tested in other systems in the hESC system. The proposal is focused on down-regulation of Chemokine (C-C motif) Receptor 5 (CCR5) in white blood cells, or ablating its expression. In addition, if time allows, similar experiments will be performed in an effort to down-regulate a second target. CCR5 serves as a co-receptor for HIV entry of R5 tropic HIV strain.

SIGNIFICANCE AND INNOVATION: This is an excellent application of hESC biology to HIV disease by a productive, innovative Principal Investigator (PI). The line of investigation is new and timely. The PI proposes to determine if an important HIV receptor can be down-regulated or knocked-out entirely in hESC, thus producing cell lines resistant to HIV infection. Such cell lines could then be differentiated into mature white blood cells for therapeutic purposes. The investigator proposes state-of-the-art gene modification methodology and, through a collaboration the PI will utilize state-of-the-art methodology for deriving mature hematopoietic cells from hESC. The approaches have been well tested in the hematopoietic system and in cultured cell lines, and therefore appear quite feasible. As appropriately emphasized by the investigator, HIV infection remains a significant problem and genetic engineering strategies to create resistant cells in infected individuals are highly desirable. The ability to expand genetically modified cells and obtain subclones offers the theoretical advantage of providing therapy with a “safer” stem cell population although the practicality of this approach and its true advantages remain to be established.

STRENGTHS: The PI is highly experienced and uniquely well-suited for this line of investigation, which would answer some important questions about both hESC responses to genetic manipulation and the utility of genetically manipulated cells in treating a global infectious disease. Establishing the methodology for this work, including the technique to modulate CCR5 expression, is important. Their collaborator also has made significant progress in obtaining hematopoietic differentiation from hESC.

WEAKNESSES: To some extent the proposal lacks innovation since it simply outlines experiments already performed with other system in the more complex embryonic stem cell system. The ability to achieve gene ablation with the proposed methodology in hESC remains to be established. There is also concern that this technique may non-specifically damage the genome with mutagenic potential. Importantly, the differences between mES cells and hESC in culture may not be fully appreciated by this investigative team.

DISCUSSION: This proposal presents a mixture of extending what this group appears to be doing already and adding the new element of the gene ablation techniques. The methodology is state-of-the-art, and the PI is a senior researcher in the field who is uniquely qualified to do the work; however there is a question of whether it is worthwhile to redo these experiments in hESCs. How applicable will hESC be to HIV studies? Reviewers felt that perhaps it might be useful work to make the cells. Regarding Specific Aim 2, one suggestion is that the PI could first screen clones for productive gene targeting events using replicative potential in vitro, and then look for those events that are non-harmful (i.e., non-leukemic).

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