The goal of this project is to develop clinically translatable methods for engineering human mesenchymal stem cells (hMSC) to serve as tumor-homing cellular carriers that will deliver a replication-competent retrovirus (RCR) vector throughout primary brain tumors (gliomas). RCR vectors expressing a prodrug activator (also known as a "suicide gene"), which converts a non-toxic "pro-drug" compound into a potent chemotherapy drug directly generated within the infected tumor cells, have recently initiated testing in Phase I/II clinical trials for suicide gene therapy of recurrent high-grade gliomas. We are examining whether MSCs can serve as producer cells for this RCR vector, and whether the tumor transduction efficiency and therapeutic efficacy of this vector can be significantly enhanced, without compromising its safety profile, hMSC-based RCR producer cells (MSC-RCR) are used as a tumor-homing mobile carrier system that releases the virus as the cells migrate toward and within tumor masses in the brain. In particular, we are comparing this MSC-RCR cell-based carrier method against conventional delivery methods by direct intratumoral injection of 'naked' virus, in subcutaneous and intracranial brain tumor models.
To date, we have accomplished our milestone tasks for Year 1, by:
- successfully developing efficient methods to transduce hMSCs with RCR vectors and thereby convert them into vector producer cells
- developing and comparing in vitro and in vivo assays to evaluate the tumor-homing migratory activity of hMSCs
- applying these assays to screen and evaluate commercially available hMSC isolates
- demonstrating that the MSC-RCR delivery system can achieve significantly more efficient transduction of subcutaneous glioma models as compared to virus by itself
- confirming that enhanced transduction efficiency by MSC-RCR achieves more rapid tumor growth inhibition, as compared to 'naked' RCR alone, when applied to suicide gene therapy in subcutaneous tumor models of human glioma
- confirming that hMSC-mediated RCR delivery does not increase vector biodistribution to normal tissues, nor incur any increased risk of secondary leukemogenesis
Interestingly, through these studies we have found considerable variability in tumor-homing migration activity and intratumoral migration activity between hMSC isolates from different sources, a finding that may have significant implications for the development of hMSC-based clinical products. We are continuing to characterize additional hMSC isolates from various tissue sources, and are preparing a manuscript to publish these results.
Furthermore, based on our favorable results as described above, indicating the enhanced efficiency of tumor transduction and growth inhibitory effects when suicide gene therapy is delivered by MSC-RCR, as compared to RCR alone, we have fulfilled the success criteria for each of our milestone tasks in Year 1, and are currently proceeding with Year 2 studies.
Reporting Period:
Year 2
Modified viruses can be used to infect tumor cells and alter the tumor cell to make anti-tumor proteins. We have developed a type of replication-competent virus that efficiently infects rapidly dividing cancer cells, but not normal brain cells. This virus is currently being tested clinically in patients with malignant brain tumors. However, to administer therapeutic virus into the brain, the virus is injected right into the center of the tumor, or in around the margins of the cavity after surgical removal of most of the tumor. Yet, human brain tumors are often found as diffusely spreading foci in the brain and may be difficult to eliminate by locally-administered replication-competent retrovirus (RCR) vectors alone. In this project, we propose to use a type of adult stem cell, called a "mesenchymal stem cell" (MSC), as a delivery system for the RCR vectors. Human mesenchymal stem cells (hMSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate through into the interior of tumor masses.
During this project period, we have established and optimized manufacturing methods to engineer hMSCs into "aircraft carriers" that release our tumor-selective RCR vectors, which we then confirmed can efficiently spread a non-therapeutic marker gene to brain tumor cells. We have further confirmed that the use of hMSCs as a cellular delivery system for RCR vectors achieves more rapid spread of the vectors through the tumor mass, as compared to injecting the virus by itself, both in tumor models implanted under the skin as well as implanted in the brain. We have also obtained initial results demonstrating that hMSC delivery of RCR vectors does not result in unwanted spread of virus to normal tissues outside the brain. This stem cell-based RCR vector delivery system, which we have so far tested and validated using a marker gene, in our current studies is now being applied to delivery of a therapeutic anti-tumor 'suicide' gene. We have also initiated discussions with the UC Davis Stem Cell Institute to develop clinical grade manufacturing processes for hMSC-based RCR vector producer cells, and with a San Diego-based biotech partner, Tocagen Inc., toward the initiation of a clinical trial to test this strategy in brain tumor patients in the near future.
Reporting Period:
Year 3
Modified viruses that have been engineered to serve as gene delivery vehicles ('vectors") can be used to infect tumor cells and alter the tumor cell to make anti-tumor proteins. We have developed a type of replication-competent virus that efficiently infects rapidly dividing cancer cells, but not normal brain cells. This virus is currently being tested clinically in patients with malignant brain tumors. However, to administer therapeutic virus into the brain, the virus is injected right into the center of the tumor, or in around the margins of the cavity after surgical removal of most of the tumor. Yet, human brain tumors are often found as diffusely spreading foci in the brain and may be difficult to eliminate by locally-administered replication-competent retrovirus (RCR) vectors alone. In this project, we propose to use a type of adult stem cell, called a "mesenchymal stem cell" (MSC), as a delivery system for the RCR vectors. Human mesenchymal stem cells (hMSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate through into the interior of tumor masses.
Through this project, we have been able to establish and optimize manufacturing methods to engineer hMSCs into "aircraft carriers" that release our tumor-selective RCR vectors, which we then confirmed can efficiently spread a non-therapeutic marker gene to brain tumor cells. We have further confirmed that the use of hMSCs as a cellular delivery system for RCR vectors achieves more rapid spread of the vectors through the tumor mass, as compared to injecting the virus by itself, both in tumor models implanted under the skin as well as implanted in the brain. We have also confirmed that hMSC delivery of RCR vectors does not result in unwanted spread of virus to normal tissues outside the brain. We have now employed this stem cell-based RCR vector platform to deliver a therapeutic anti-tumor 'suicide' gene, and we have shown that stem cell-mediated vector delivery results in longer survival compared to delivery of the virus by itself. We have also initiated discussions with the UC Davis Stem Cell Institute to develop clinical grade manufacturing processes for hMSC-based RCR vector producer cells, and with a San Diego-based biotech partner, Tocagen Inc., toward developing a clinical trial to test this strategy in brain tumor patients in the near future.
Reporting Period:
NCE (Year 4)
Gene therapy can be used to alter the genome of cancer cells and induce them to make anti-tumor proteins. We have developed a highly efficient gene delivery vehicle (known as a “vector”) derived from a modified virus, which efficiently spreads though brain tumors and infects and permanently alters the genome of cancer cells, but does not infect normal brain cells. This modified virus, called a “replication-competent retrovirus (RCR) vector”, is currently being evaluated in clinical trials on-going at multiple sites throughout California to treat patients with malignant brain tumors, with highly encouraging results. However, to administer the therapeutic virus into brain tumors, the virus is injected directly into the center of the tumor, or around the margins of the cavity after surgical removal of most of the tumor. Yet, human brain tumors often diffusely spread into the surrounding normal brain tissue, and may be difficult to eliminate with a locally-injected RCR vector by itself. Therefore, in this project, we evaluated the use of a type of adult stem cell, called a "mesenchymal stem cell", as a delivery system for RCR vectors. Human mesenchymal stem cells (hMSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate throughout the interior of tumor masses.
Through this project, we have established and optimized manufacturing methods to engineer hMSCs into "aircraft carriers" that release our tumor-selective RCR vectors, which we then confirmed can efficiently spread both a non-therapeutic “reporter gene”, as well as a therapeutic “suicide gene” to brain tumors. We have further confirmed that the use of hMSCs as a carrier system for delivery of RCR vectors results in more rapid spread of the vectors through the tumor mass, as compared to injecting the virus by itself, in human brain tumor models implanted both under the skin as well as in the brain. We have also confirmed that, when this hMSC -based RCR vector delivery system is employed to deliver an anti-tumor 'suicide' gene, the faster spread of the virus delivered by the stem cell carrier translates into more rapid shrinkage of tumors implanted under the skin, and prolongs survival in intracranial brain tumor models. In the final project period, we have also obtained results demonstrating that hMSC delivery of RCR vectors injected into intracranial brain tumors does not result in unwanted spread of virus to normal tissues outside the brain. We have initiated discussions with the UC Davis Stem Cell Institute to develop clinical grade manufacturing processes for hMSC-based RCR vector producer cells, and with a San Diego-based biotech partner, Tocagen Inc., toward the initiation of a clinical trial to test this strategy in brain tumor patients in the near future.
Grant Application Details
Application Title:
Stem cell-based carriers for RCR vector delivery to glioblastoma
Public Abstract:
Modified viruses can be used to infect tumor cells and alter the tumor cell to make anti-tumor proteins. Most researchers use virus that can infect and modify the tumor cell it enters, but can not make more of itself to infect additional cells surrounding the original infected cell. This type of virus is called replication-incompetent virus. Use of replication-incompetent virus is considered safe because no additional virus, which potentially could get out of control, is generated inside of the tumor. However such therapies have been shown to have only limited beneficial effects, presumably because too many tumor cells never get infected.
Newer approaches investigate the use of replication-competent viruses to achieve highly efficient gene transfer to tumors. A successfully transduced tumor cell itself becomes a virus-producing cell, sustaining further transduction events even after initial administration. We propose here to use a type of replication-competent virus that only infects dividing cells and therefore will infect the rapidly dividing cancer cells but not normal brain cells.
The use of replication-competent virus is potentially more risky but is well justified in clinical scenarios involving highly aggressive and rapidly progressing metastatic tumor growth in the brain. To administer therapeutic virus into the brain, the virus is injected right into the center of the tumor. Yet, human brain tumors are often found as diffusely spreading foci in the brain and may be difficult to eliminate by locally-administered replication-competent retrovirus (RCR) vectors alone.
In this study we propose to use a type of adult stem cell called a "mesenchymal stem cell" (MSC) as a delivery system for the RCR vectors. Mesenchymal stem cells (MSCs) have been shown to have natural tumor-homing abilities, and can migrate to tumor foci and penetrate through into the interior of tumor masses. We propose to engineer them into "aircraft carriers" that release tumor-selective viruses, which can then efficiently spread suicide genes from one cancer cell to another in multiple tumor foci in the brain.
Statement of Benefit to California:
This research is based on a solid foundation that combines two innovative technologies for the treatment of primary brain tumors, particularly glioblastoma multiforme (GBM) the most malignant form of brain tumor, which afflicts men, women, and children in California and elsewhere. Each of these technologies has been approved separately by FDA for clinical testing in humans: human mesenchymal stem cells (MSCs), and replication-competent retrovirus (RCR) vectors.
MSCs have been reported to exhibit a natural ability to migrate to solid tumors and penetrate into the tissue mass. Once inside a tumor, RCR vectors can spread selectively in the cancer cells and their replication can keep up with their uncontrolled proliferation, and their ability to integrate themselves into the cancer cell genome allows them to permanently "seed" tumor cells with therapeutic genes.
Here we propose to utilize the natural tumor homing ability of MSCs to deliver RCR vectors into brain tumors. This "virus vs. cancer" strategy takes advantage of the amplification process inherent in the spread of virus from cell to cell, and by using MSCs to initiate the virus infection efficiently in brain tumors, represents an approach that will have the potential to effectively treat this poor prognosis disease.
If successful, clinical application of this strategy can be implemented by an "off-the-shelf" mesenchymal stem cell (MSC) primary cell lines that have been pre-characterized for their tumor homing ability and virus production capability, and can be offered to patients without requiring an invasive procedure to harvest their own stem cells. Furthermore, this represents a treatment that could potentially be administered through a needle, thus making it unnecessary for patients to undergo major neurosurgical procedures entailing craniotomy at an advanced medical center. Hence this research could lead to a novel treatment approach that would particularly address the needs of brain tumor patients in California who are underserved due to socioeconomic and geographic constraints, as well as the elderly who are poor-risk for surgical interventions.
