Equipping Stem Cells

Both embryonic and adult stem cells can be expanded in vitro and stored for long periods of time for later clinical usage. These cells can be transduced with numerous viral vectors to express transgenes which code for prodrug-activating enzymes, inducers of apoptosis, differentiating agents, cell cycle modulators, anti-angiogenesis factors, immune-enhancing agents, and oncolytic factors. By taking advantage of stem cells’ preferential migration toward sites of pathology, they can be made to engraft to malignant tissues and continually secrete their transgene product. A remarkable characteristic of stem cells is their ability to migrate to tumors regardless of size, anatomic location, or tissue of origin.(19) Different types of therapeutics are best delivered by various cell types that are engineered for treatment of a specific disease. As such, there are innumerous choices of stem cell type and transgene combinations. Yet this general strategy has been implemented by various groups, and a selection of the most promising methodologies is discussed below.

Of initial concern for treatment is the methodology used for transduction of the desired stem cells. Usage of adenoviruses and retroviruses is most common, however, some groups have also used adeno-associated viruses, herpes simplex virus 1, and human immunodeficiency virus 1.(20)(21)(22) The rationale for choosing one method for transduction over another involves a number of factors including genomic integration, compatibility with the given cell type, and the immune response they and their products illicit. For instance, usage of retroviral transduction allows for long-term transgene expression due to integration into the host DNA. Alternatively, usage of an adenovirus for transfection allows for transient but high-level transgene expression due to lack of host DNA integration.

There are various transgene products which must then be chosen for the particular application. These generally fall into two categories: prodrug activating agents and therapeutics which directly disrupt tumor cells. The usage of prodrug activating agents is attractive because both the transgene product and administered prodrug are designed to have minimal toxicity in a patient. The assumption with their use however, is that locally activated drugs will not travel away from the site of pathology and affect surrounding tissues significantly. A recent study using neural stem cells transfected with cDNA which encoded for rabbit carboxylesterase, an enzyme which activated the prodrug CPT-11, recorded high concentrations of the activated prodrug in disseminated neuroblastoma tumors in mice, but little to no activated prodrug elsewhere.(23) This is indicative that usage of stem cells equipped with transgenes which express prodrug activating products may indeed provide very selective drug localization.

The prodrug activation avenue has been adopted by other groups as well with large degrees of success. The gene coding for cytosine deanimase, which converts the prodrug 5-FC to 5-fluorouridine and which then inhibits DNA and RNA synthesis has been transduced into neural stem cells via retrovirus by several groups to treat mice with medulloblastomas. These groups have recorded extended survival versus control as well as up to a 76% reduction in tumor volume.(24)(25) An alternative prodrug activating enzyme, rabbit carboxylesterase, has also been transduced into neural stem cells via an adenovirus. Subsequent activation of the prodrug irinotecan has had extremely promising results in mice bearing disseminated neuroblastoma. One group found that intravenous administration of transduced stem cells produced greater than one year survival in 90% of the mice.(26)

Of the transgene products which function to directly interfere with tumor cell functionality, the most promising have been tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the human metalloproteinase-2 fragment PEX, and interleukins. TRAIL has been transduced into neural stem cells using an adenovirus and has elicited a >80% reduction in mouse gliomas. TRAIL is also attractive because it has been shown to have minimal effect on the surrounding tissue.(27) PEX was similarly transduced into neural stem cells using a specialized PTracer-BsdPEX vector and was shown to reduce mouse glioma volume by a comparable 90%. (28)

Finally, interleukins have provided the most interesting results. One group utilizing retrovirus-mediated transduction of neural stem cells to create a cell line which expressed interleukin-4 injected these cells into mice with established syngeneic brain glioblastomas. After 90 days, 5 out of 6 of the treated mice were still alive as compared to none in the untreated group. They also noted that mice injected with neural stem cells that had not been transduced with the interleukin 4 gene exhibited a greater survival rate than the control group. This indicates that stem cells alone may have inherent pro-apoptotic properties toward glioblastomas.(29) A similar approach to the same problem was taken by another group who utilized neural stem cells transduced with the interleukin 12 transgene. They similarly found that mice treated with these stem cells exhibited markedly longer survival times than the control group. They also noted that mice treated with interleukin 12 secreting neural stem cells demonstrated significantly increased infiltration of CD4+ and CD8+ T-cell into tumor microsatellites