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Regulatable genetic modifications of HESC for in vivo imaging and safety monitoring of implanted HESC: applications to neurodegeneration models

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01110
Funds requested: 
$952 413
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells (HESC) can be differentiated into any cell of the body. This will allow HESC to be used to replace diseased or damaged cells. The brain is made up of mostly non-dividing cells; thus, upon brain cell death, there are no new cells that can take up the role of the diseased or damaged cells. HESC are thus of great importance for the future replacement of brain cells damaged in neurodegenerative disorders such as Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), or multiple sclerosis. When sufficient brain cells have been killed by the disease process and no functional capabilities can be sustained, disease symptoms ensue. If cells in the substantia nigra die, patients develop PD; if cells from the basal forebrain and cerebral cortex die, they will develop AD, and if motor neurons from the anterior horn die, they will develop ALS. Fortunately, HESC can be differentiated into dopaminergic neurons (like those of the substantia nigra), cholinergic neurons and cortical neurons (such as those dying in AD), and into motor neurons (affected by ALS). Implantation of neurons of the right type into the diseased region of the brain could potentially overcome the severe symptoms of these diseases. There are two challenges when HESC are implanted into diseased brains. One is to be able to track the implanted cells; i.e. are they staying within the implanted region, or are they moving away? Secondly, will HESC remain as differentiated cells, or will they form a tumor or teratoma. To address these issues we will develop two viral vectors, one derived from HIV and called a lentivirus (LV), and one derived from adenovirus, called a high-capacity adenovirus (HC-Ad). Both vectors have been engineered to remove their toxic and pathogenic genes, and are safe for use in animals and humans. The system will work in the following manner: LV will express genes in both non-dividing and dividing cells, while HC-Ad will sustain expression in non-dividing cells, but will be lost from dividing cells. HESC will be differentiated before implantation; to be of therapeutic use they should remain differentiated, and thus, non dividing. If they do, we will detect this as imaging signals originating from both vectors. However, it will be crucial to monitor in humans whether suddenly these cells start growing into a stem cell tumor, also known as teratoma. Should this happen, we will detect an increasing signal from the LV, but will lose the signal from the HC-Ad. Importantly the engineered HESC will have a gene encoded within their genome which will allow us to kill them should they become cancerous. In summary, we propose to develop a novel tool to track the location of implanted HESC in the brain, and their growth patterns. The system will be engineered to allow imaging in experimental animals and humans, thus, being a valuable translational tool as HESC move from the bench to therapies.
Statement of Benefit to California: 
The promise of harnessing human embryonic stem cells for the treatment of human disease is being stimulated in great measure by the California Initiative in Regenerative Medicine. The graying of California’s population is given by the fact that this is the most populous state in the Union, and thus, subjected to the progressive ageing of our population, as seen throughout the USA. In addition, due to its very favorable climate, California is increasingly attracting retirees to its midst. As a consequence it is highly likely that due to these demographic factors, California will experience a continued increase in the incidence of serious chronic neurodegenerative disorders. Many of the diseases of old age sadly affect the structure and function of the brain. HESC’s promise is to develop cells that will replace cells affected by these chronic, progressive diseases. Although sometimes diseases like Parkinson’s, Alzheimer’s or Lou Gehrig disease can affect patients in their late thirties or early forties, most of the patients affected by these diseases are 60 years of age or older. California’s population is expected to increase by 172% by 2040, with the greatest growth being among those aged 85 and older. Their numbers are expected to grow by 200% by 2040. Thus, it is expected that by 2040 the ratio of the elderly to adults under age 65 will have increased by 80%! It is expected that by 2010 there will be close to 500,000 Californians with Alzheimer’s alone, and could rise to 1,000,000 by 2040. Added to other chronic neurodegenerative diseases with increased incidence in the aged, new therapies are urgently needed. The proposed application will develop unique novel tools and technologies to track the location and fate of implanted therapeutic HESC in the brains of affected patients. In addition, the novel tools will have safety elements that will allow to monitor whether implanted HESC become tumoral, and if so, to eliminate them. In summary, work proposed in this application will provide a strong platform for the development of effective and safe HESC-based therapies for devastating chronic brain diseases. Further, the HESC and the novel viral vectors to be used in this proposal have already been generated by our team in the State of California, thus, taking advantage of the intellectual and technological know-how developed in our State.
Review Summary: 
This proposal focuses on the development of novel technologies to track transplanted cells and to allow noninvasive detection of teratoma formation following transplantation. Specifically, the applicant proposes to infect cells with both an integrating lentiviral vector (LV) and a non-integrating high-capacity adenoviral vector (HC-Ad). The integrating lentivirus will be retained in non-dividing and in dividing cells, whereas the non-integrating adenovirus will be retained in non-dividing cells but lost from dividing cells such as tumor cells. These viruses will be engineered to express markers that can be detected using various imaging techniques, including the in vivo imaging methods positron emission tomography (PET) and magnetic resonance imaging (MRI). The principal investigator (PI) proposes to test the system in a rodent model. Human embryonic stem cells (hESCs) containing these viruses will be transplanted into rat brains, and the investigators will try to assess survival, migration, and possible proliferation of these cells using PET, MRI and bioluminescence. Finally, investigators will validate the utility of these novel technologies by transplanting hESC-derived dopaminergic precursor cells transduced with these two vectors into a mouse model of Parkinson’s disease. They plan to assess the movement and cell division of these dopaminergic cells in vivo using PET and MRI, and then confirm their imaging results with post mortem immunohistochemistry. They also plan to evaluate animal motor behavior for functional effects of the transplanted cells. The reviewers felt this was an innovative and ingenious proposal addressing a significant roadblock in stem cell biology. However, they raised some questions about its feasibility and would have liked to see more preliminary data demonstrating proof-of-principle. The reviewers praised the quality of the research team but were concerned that the PI might be somewhat over-committed. The reviewers agreed that this proposal could have a broad impact in the field. Teratoma formation by transplanted hESC-derived cells is a tremendous potential risk, so this proposal addresses an important roadblock. If successful, the proposed technology would allow for non-invasive monitoring of the uncontrolled proliferation that leads to tumor formation. It would also provide, through suicide genes, a method for eliminating such proliferating cells. Reviewers questioned whether dual virus treatment will ever be practical for clinical applications but acknowledged that viral technologies are improving rapidly. They felt that the tools generated in this proposal would be valuable even if they were restricted to animal studies. The reviewers raised concerns about the feasibility of this proposal and would have liked to see more preliminary data confirming the validity of the approach. They noted that it was unclear how many divisions would be required to dilute out the HC-Ad markers. If the number were high then the value of the technique in detecting early tumors would be limited. This issue could be addressed with preliminary data in vitro. Reviewers were also concerned that HC-Ad expression may be lost in a matter of weeks even in the absence of cell division, and would have liked to see data addressing its stability in differentiated neurons. One reviewer would have liked to see data using ferritin as an MRI marker, as this is the only HC-Ad marker of current clinical usefulness in patients. Another reviewer raised a few minor concerns with the experimental design and data presentation. The applicants describe implanting a half million cells into the target region of the striatum and using a basic amphetamine-induced rotational test to monitor behavioral changes after implantation. If the dopamine neuron line is unsuccessful in improving amphetamine-induced rotational behavior, the back-up plan is simply to administer larger numbers of cells. However, the reviewer noted that many recent investigations of transplanted human fetal cells point to an overabundance of dopamine as causal to the generation of dyskinesias in humans and in animal models. Thus, the investigators would be well served to instead consider a more appropriate implantation site as a back-up plan, like the substantia nigra, and also to use more sophisticated behavior tests such as forelimb reaching. This reviewer also noted some ambiguities in the preliminary data, including in Figure 6 where five of the images are simply identified as substantia nigra immunoreacted for TH and fluorogold. One of these images shows positive cells, but without identifying what the colors mean or what the other images might be, making the figure unhelpful. Likewise, some of the images in Figure 7, such as C and D, which are purported to demonstrate a protective effect of GDNF do not show higher numbers of cells in comparison to the vehicle, although the bar graph below does. The reviewers were enthusiastic about the quality of the research team and felt that they are clearly qualified to carry out the proposed research. Overall this is an innovative proposal from an excellent team that addresses a significant roadblock in stem cell research. However the reviewers raised some concerns about feasibility and felt that the preliminary data could have better demonstrated proof-of-principle. PROGRAMMATIC REVIEW A motion was made to move this proposal from Tier 3 to Tier 2 and recommend for funding if additional funds become available - the motioner argued that this is an innovative proposal that could have a substantial impact and bring stem cell therapies for neurodegenerative disease closer to the clinic. Panel members discussed the important issue of whether HC-Ad would be stable in the cell long enough to be useful. One member asserted that HC-Ad will stably express in neurons for as long as a year. Another panel member cited the problem of finding the rare cells that have lost the marker and asked if imaging technologies would be capable of detecting the indicated markers. Reviewers seemed to think they would. Panel members were divided on the potential clinical applicability with some arguing that it would be very difficult to get approval to put 2 vectors into humans and others suggesting that it would be possible with significant advances in vector technology. Overall, reviewers felt that the programmatic reasons for funding the proposal were strong enough to justify the scientific uncertainties regarding feasibility. The motion carried.
Conflicts: 

© 2013 California Institute for Regenerative Medicine