Liver Disease Team

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01490
ICOC Funds Committed: 
$0
Disease Focus: 
HIV/AIDS
Immune Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
Public Abstract: 
Because there is still considerable morbidity and mortality associated with the process of whole liver transplantation, and because more than a thousand people die each year while on the liver transplantation list, and tens of thousands more never get on the list because of the lack of available livers, it is evident that improved and safer liver transplantation would be valuable, as would approaches that provide for an increased number of transplantations in a timely manner. A technology that might address these issues is the development of a human liver cell line that can be employed in liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics. The objective of this proposal is to focus a team effort to determine which differentiated hESC will be the most effective liver-like cells in cell culture and in animal studies, and to then use the best cells in clinical trials of cell transplantation in patients with advanced liver disease. In the proposed studies, the team will differentiate hESC so that they act like liver cells in culture. Once it has been established that the cells are acting like liver cells by producing normal human liver proteins, and that they do not result in tumors, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. One of the ways cells can be evaluated is to label the cells which will provide a means to monitor them with various imaging systems. The intent in these studies is to determine which will be the most effective cells to use in human clinical trials. Once this is determined, the best cells can then be employed in human patients. If the studies are successfully undertaken, we will have established a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
Statement of Benefit to California: 
In California, as in all parts of the US, there are not enough livers available for transplantation for all the people who need them. The result is that many more people die of liver failure than is necessary. One way to improve this situation is the transplantation of liver cells rather than whole organ transplantation. We are attempting to develop liver cell lines from stem cells that will act like normal liver cells. If the cells that we develop function well and do not act like cancer cells in culture, the cells will be assessed in clinically-relevant models using techniques that can then be adapted to future human clinical trials. In our studies, we will compare human embryonic stem cells with other stem cells to determine which will be the most effective cells to transplant into people. Finally, we will employ the best cells in clinical trials in humans. If the studies are successfully undertaken, we will have established a clinically useful and viable liver cell line that could be used to repopulate an injured liver in a safer and less expensive manner than with whole liver transplantation. Moreover, all people who have liver failure or an inherited liver disease could be treated, because there would be an unlimited supply of liver cells.
Progress Report: 
  • During the first year of the project, we have made significant progress in meeting the first milestone of the project: Defining the final process of genetically modifying hematopoietic stem/progenitor cells (HSPC) (Item #14, Milestone M3 of Gantt chart). In addition, initial effort has started in Phase II Scale-up/Pre-clinical testing (G15) and more specifically, in hematopoietic stem/progenitor cell processing development (G16).
  • Some 10 years ago it was discovered that patients homozygous for a natural mutation (the delta 32 mutation) in the CCR5 gene are generally resistant to HIV infection by blocking virus entry to a cell. Building on this observation, a study published in 2009 reported a potential "cure" in an AIDS patient with leukemia after receiving a bone marrow transplant from a donor with this delta 32 CCR5 mutation. This approach transferred the hematopoietic stem/progenitor cells (HSPC) residing in the bone marrow from the delta 32 donor, and provided a self-renewable and lifelong source of HIV-resistant immune cells. After transplantation, this patient was able to discontinue all anti-HIV drug treatment, the CD4 count increased, and the viral load dropped to undetectable levels, demonstrating an effective transplantation of protection from HIV and suggesting that this approach could have broad clinical utility.
  • But donors with the delta 32 CCR5 mutation are not generally available, and so how could we engineer an analogous CCR5 negative state in human HSC to be used for bone marrow transplantation, including a patient’s own HSPC? A potential answer comes from zinc finger nucleases (ZFNs) which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an error-prone DNA repair process. This modification of the cellular DNA is permanent and can fully block gene function. Recently, ZFNs have been shown to inactivate CCR5 in primary human CD4 T cells, allowing them to preferentially survive and expand in the presence of HIV. A human clinical trial evaluating this approach is on-going, in which patient T cells are re-infused after ZFN-treatment to block CCR5 expression and possibly provide an HIV-resistant reservoir of CD4 T cells.
  • This CIRM Disease Team proposed an approach to modify a patient’s own HSPC to circumvent the need to find matched donors that carry the delta 32 CCR5 mutation and yet provide a renewable and long-lasting source of HIV-resistant cells. Testing of this concept is proposed in selected AIDS lymphoma patients who routinely undergo HSPC transplantation. During the second year of this project, the disease team has made considerable progress and met all the project milestones for year 2. More specifically, the team developed an optimized procedure for efficiently introducing the CCR5-specific ZFNs in HSPC. We showed that these modified cells function normally and retain their “stemness” in tissue culture systems. We also showed these modified cells can be transplanted into mice to reconstitute the immune system. Given HSPC are long lasting stem cells, we have been able to stably detect these cells in mice for over 3 months post-transplantation. The team is in the process of scaling up the cell production procedures to ensure we can generate CCR5-modified HSPC at clinical scale. We are also moving ahead with the remaining pre-clinical safety and efficacy studies required before initiating a clinical trial.
  • 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.

© 2013 California Institute for Regenerative Medicine