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Developing an Effective Stem Cell Gene Therapy to Treat Human Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00186
Funds requested: 
$499 999
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The human body functions because there are specific proteins (enzymes) that perform all the work (chemical reactions) necessary to allow mechanical movement, eating, reproducing and eliminating wastes. Enzymes are produced by specific genes. Each person can have slightly different enzymes produced by slightly different genes. Sometimes these changes provide advantages, sometimes these changes cause dis-advantages. Essentially all diseases can be linked to the interference of one or more enzymes in regard to performing its chemical reaction. Human medicine has been based on drugs, which are chemicals that affect the activity of enzymes. By increasing or decreasing the activity of enzymes, drugs can reduce and (sometimes) eliminate the deleterious effects of diseases. A potential problem with many drugs is that they commonly are associated with unwanted side-effects and their potency can be reduced by acclimation of the body’s response to that particular drug. With the advances in understanding the human body, it is now possible to cure diseases by altering the expression of genes to affect the activity of specific enzymes. The advantages of “gene therapy” are specificity to the established target, long-term duration of treatment and reduced chances of side-effects. While gene therapy has been proposed as a method to treat many human diseases having devastating effects, there are limited examples of successful outcomes. We are now at a cross-roads in our knowledge of being able to learn how to develop successful gene therapies for treating human disease. One of the major obstacles is having the appropriate vehicle to deliver the gene therapy to the patient. We have developed a simple method to deliver therapeutic genes to mice using stem cells that will repopulate the liver in the form of liver macrophages (i.e. Kupffer cells). Kupffer cells are immunologic cells that normally have a long lifetime, reside on the surface of liver cells and protect the body from bacteria that enter the body via the intestine. A safe drug, used to visualize arteries when irradiated with x-rays, is eaten by Kupffer cells causing them to be rapidly eliminated and replaced by new Kupffer cells which are derived from stem cells. By switching the stem cells used for replacing Kupffer cells with stem cells carrying a specific therapeutic gene, we have shown we can reduce heart disease in mice in manner that is safe, without any toxic side effects and long-term (up to a year). The goal of this research is to further develop the Kupffer cell gene transfer technique for treating human disease. By developing an efficient means to use embryonic stem cells as the vehicle, we will be able to provide a safe, versatile, reversible and effective means to provide gene therapies for many different type of human diseases including: diabetes, infectious disease, heart disease, liver failure and metabolic deficiency diseases.
Statement of Benefit to California: 
During the past 20 years, scientists (many residing in California) have discovered how to identify genes, how to identify the functional role of the proteins made from these genes and how to identify if individuals have mutations in genes that affect function. In addition to providing patients with an understanding why they may have a particular disease, these advances have provided new clues that may be useful in designing a new way to treat diseases called gene therapy. Gene therapy has the potential to cure many diseases for which drugs have not been developed or have been shown to have bad side-effects. Because of its many potential benefits, scientists have spent ~20 years designing methods to administer gene therapies that are safe and effective. So far, successful amelioration of human disease via gene therapy has been a rare occurrence. We have developed a new method to administer gene therapy to mice. This method is safe and effective (treating mice with this gene therapy reduced heart disease in mice by 50%). We wish now to further develop this method so it can be used to treat humans. The goal of this research to develop this method and show that it is safe and effective in mice. Achieving this goal will justify it being further developed for use in humans in treating many diseases including: diabetes, heart disease, liver disease, several metabolic deficiency diseases, several forms of malignant diseases involving plasma cells, infection diseases and diseases associated with impaired immunity. This investment by CIRM in supporting this research has the potential to provide new, safe and effective gene therapies to the citizens of California.
Review Summary: 
SYNOPSIS: Two specific aims are purposed: 1) to examine the hypothesis that genetic ablation of Txnip will enhance the survival of ESC and their ability todifferentiate into HSCs, Kupffer cells and resident macrophages; and 2) to examine the therapeutic potential of this gene transfer method. The applicant will examine how reconstitution of bone marrow and hematopoietic stem cells having genetic ablation of Txnip affects cardiac repair following ischemic injury, liver repair following toxic injury and protection of pancreatic β-cells from apoptosis induced by insulin resistance and hyper-secretion stress. SIGNIFICANCE AND INNOVATION: The investigator purposes to explore the ability to derive monocytes and macrophages from murine embryonic stem cells for utilization in reconstituting the Kupffer cell mass in murine transplant recipients. In prior work he has shown that hematopoietic stem cells expressing the transgene encoding the atherosclerosis-protective gene, paraoxonase1, have the potential for reducing the formation of atherosclerosis by 50% in a murine model of human homozygous familial hypercholesterolemia. The basic strategy used in those experiments was to transplant lethally irradiated mice with hematopoietic stem cells encoding the PON1 transgene. After hematopoietic reconstitution had occurred, the mice were given gadolinium chloride which depletes Kupffer cells by inducing apoptosis. The Kupffer cell mass is reconstituted with macrophages derived from the transplanted hematopoietic stem cells. The investigator has also developed evidence that ablation of the gene encoding the pro-inflammatory, pro-apoptotic thioredoxin interacting protein (Txnip) enhances cell viability, increases cell replication and decreases apoptosis. The proposed experiments are highly innovative and yet based on prior experimental work by this investigator. He proposes to use genetically modified mouse strains to explore the ability to generate engraftable macrocytes from human embryonic stem cells in vitro. Gene delivery by this method would indeed have significant potential impact for the treatment of human disease. An innovative aspect is related to understanding the mechanisms that relate to the role of a pro-apoptotic interacting protein gene in promoting the survival of hematopoietic stem cells. This study will determine if the loss of this gene will enhance the stem cell functions of bone marrow derived HSCs in multiple forms of injury, largely based on precepts related to the assumption that these blood derived cells can exert a beneficial effect on other organ systems, presumably via repopulation of the tissue specific monocytes analogous to Kupffer cells in other organ systems. The approach would require both a gene and cell based transfer, and the optimal clinical approaches for the former (gene therapy) with regards to vector are yet to be established. STRENGTHS: -Familiarity of the investigator with the purposed techniques. The investigator is experienced and has published extensively in this field, and has an excellent track record. -Extensive prior data indicating that Kupffer cell mass can indeed be replaced by the strategies purposed by the investigator. -The work is feasible, and the preliminary data are solid for much of the work. -Established collaborations with experienced investigators having specific expertise in aspects of the purposed project. -The scientific environment is strong for this work. WEAKNESSES: - The initial 2 experiments proposed to address specific aim 1 seem very predictable: GdChl3 treatment was found to have increased RNA expression of IL-1, IL-3, M-CSF, GM-CSF and TNFα. Culture medium containing these cytokines was found to induce differentiation of embryonic stem cells to monocyte-macrophage progenitors. Since these results have already been obtained, what new will be learned? - In addressing Specific Aim 2, the investigator proposes to use bone marrow hematopoietic stem cells to achieve hematopoietic reconstitution with bone marrow cells having genetic ablation of Txnib. However, in the proposed experiments, it is not clear whether he will use animals reconstituted with genetically modified hematopoietic stem cells or whether macrophages/monocytes derived from genetically modified embryonic stem cells in vitro will be utilized in an attempt to prevent or ameliorate tissue damage. The later would seem to be more directly relevant to the goals of the purposed experimentation. - The overall assumption that HSCs, via their effects on monocytes, can exert a significant, clinically relevant effect has inherent difficulties. Mononcytes can fuse with other cell types and some of the observed transdifferentiation might reflect this point. - The relevance of the experiments in other organ systems, as in the beta cells in the pancreas, seem to be driven by their potential clinical importance if they were to succeed, but there is little data to support this concept at this time. - As the studies do not incorporate studies on human ES cells, one of the major points of reponsive to the RFA has not been addressed. DISCUSSION: This proposal is not responsive to the RFA.
Conflicts: 

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