Year 3

It is well known that infection with HIV-1 requires a protein called CCR5, and persons with a natural mutation in this gene (CCR532) are protected from HIV/AIDS. Everyone has two copies of the CCR5 gene, one inherited from their mother and one from their father. People with both copies of CCR5 mutated (CCR532/ CCR532) are highly resistant to becoming infected with HIV-1. If only one copy is abnormal (CCR5/ CCR532), infection can occur but progression of the infection to AIDS is delayed. The only clear cure of HIV-1 infection occurred in a patient with leukemia who received a blood stem cell transplant from a tissue-matched donor whose cells carried the double mutation CCR532/CCR532. After transplantation, this patient was able to stop all anti-HIV medicine, the immune system improved, and the level of HIV-1 in the blood dropped to undetectable levels. Even after more than 4 years off anti-HIV medicine, the patient is considered cured, as there is no evidence of an active HIV-1 infection.

This Disease Team proposes to treat blood stem cells from an HIV-1 infected person with a protein that can mutate the CCR5 gene, and then transplant these same cells back into the patient to try and reproduce the effects of the CCR532 mutation by providing a renewable and long-lasting source of HIV-1 resistant cells. This will circumvent the need to find a stem cell donor who happens to carry the CCR532/ CCR532 mutation and is a suitable “perfect match” for tissue transplant. The proteins that will be used in this treatment are called Zinc Finger Nucleases (ZFNs). Preliminary results in mice transplanted with ZFN-treated blood stem cells have shown that the modified cells are functional and produce CCR5 mutant progeny cells – including CD4 T cells that are the natural target of HIV-1. Importantly, after HIV-1 infection, the mice demonstrated reduced viral loads, maintenance of CD4 T cells in peripheral tissues, and a powerful survival advantage for the CCR5-negative cells [Holt et al., Nature Biotechnology 2010; 28: 839-47]. These data support the development of this ZFN approach to treat HIV-1 infected patients by first isolating the subjects own blood stem cells, modifying them using CCR5-specific ZFNs, and then re-infusing them back into the patient to thereby reconstitute the immune system with CCR5-mutant, HIV-1 resistant cells. The Disease Team assembled to accomplish this goal has expertise in stem cell technology [City of Hope], HIV-1 infection in pre-clinical mouse models [University of Southern California], and in ZFN-based clinical trial development [Sangamo BioSciences].

In the first two years of study, the Disease Team focused on the use of an existing delivery technology for introducing the ZFNs into blood stem cells. This approach used a type of gene therapy vector called an adenoviral vector, which had been previously used in early stage investigational clinical trials for the modification of patients’ T cells. During this phase of the project, the Disease Team was able to establish a method that allowed the large scale manufacture of ZFN-modified blood stem cells under conditions suitable for a clinical trial. These results were recently published [Li L. et al. Molecular Therapy; advance online publication 16 April 2013]. In year 3 of the study, the Disease Team developed a new method for delivering the ZFNs to the blood stem cells using messenger RNA (mRNA, or SB-728mR). Using a process called electroporation, in a technique that involves exposing a mixture of the blood stem cells and the SB-728mR to a transient electrical field, efficient mutation of the CCR5 gene was achieved. These cells were able to be transplanted into mice, where they engrafted and differentiated to generate human immune cells carrying mutated CCR5 genes. This mRNA-based approach has proven to be robust, well-tolerated and eliminates all viral vector components from the manufacturing process. Thus, electroporation of SB-728mR has now been chosen to move into clinical-scale manufacturing and to support our proposed clinical trial. In Year 4 of the study, the Disease Team will complete the necessary studies to demonstrate the safety of these modified blood stem cells, and submit the required federal and local regulatory documents to support the Phase I clinical trial of this new drug.