Genetically Altered Hematopoietic Cells Used in Transplant Research

SEATTLE--Genetically modified hematopoietic cells are being usedto protect healthy stem cells from toxic drugs in early clinicaltrials and, in cell lines, to sensitize cancer cells to toxicdrugs and to induce leukemia cells to revert to a normal phenotype,Albert Deisseroth, MD, PhD, said at a symposium held in conjunctionwith the American Society of Hematology meeting. Dr. Deisserothis associate director for clinical research, Yale University CancerCenter.

In one such protocol, using a retroviral vector, the Yale researcherstransduce the human multidrug-resistance gene (MDR-1) into patientstem cells that have been purified via a monoclonal antibody columntechnique. These genetically altered cells are returned to thepatient, who then receives paclitaxel (Taxol) therapy.

Studies using this technique are now underway at the NIH, M.D.Anderson, and Columbia University, in nine ovarian cancer patientsand nine breast cancer patients, Dr. Deisseroth said. Thus far,the studies have shown rapid recovery of hematopoietic function,and the cells that have repopulated the marrow do carry the transducedMDR-1 construct.

"We're in a position now," Dr. Deisseroth said, "totest whether post-transplant chemotherapy can select for geneticallymodified cells; in other words, whether the MDR-1-carrying cellswill thrive after these patients are treated with Taxol, thusreducing the cytopenia that usually complicates chemotherapy.If so, the result would be chemoprotection."

Dr. Deisseroth also theorized that retroviral vectors in hematopoieticcells might be used to help overcome the problem of residual cancercells contaminating transplanted marrow. The approach involves"chemosensitizing" the bone marrow cells, via genetictransduction, in such a way that the cancer cells are destroyedby chemotherapy while normal cells remain unharmed.

The Yale research team used an adenovirus vector linked to a transcriptionunit that codes for cytosine deaminase, a bacterial gene not presentin mammalian cells. This enzyme is able to deaminate the innocuousdrug 5-fluorocytosine (5-FC), converting it into the highly toxicdrug, 5-fluorouracil (5-FU).

The key point of this system, Dr. Deisseroth said, is that theadenovirus vector binds avidly to an integrin receptor on neoplasticcells but does not bind avidly to early hematopoietic cells. "Thus,the vector has a specific tropism for the very cells we want todestroy," he said.

Another advantage to this particular vector is that, once it isinside the cell, its transcription unit can be expressed withoutintegration into the host cell's DNA--thus, nonproliferating cellsare as susceptible as rapidly dividing ones.

The study found that, in culture, unmodified breast cancer cellswere relatively unaffected by treatment with 5-FC, but 100% ofthe cytosine deaminase gene-modified breast cancer cells werekilled by the same 5-FC concentration.

"We're trying to develop this genetic chemosensitizationas an ex vivo, in vitro modification protocol," Dr. Deisserothsaid. "But obviously, being able to manipulate populationsof somatic cells with this type of vector may prepare us for anapproach to systemic gene therapy."

Genetic Transduction

Dr. Deisseroth and his colleagues are also exploring the possibilityof using genetic transduction to induce cancer cells to revertto a normal phenotype. The goal is to disrupt the specific fusionproteins that arise from the translocations seen in leukemia.

The team has tested a genetic suppressor element (GSE) that affectsthe polymerization of the BCR/ABL p210 fusion protein. When thisGSE was transduced via a retroviral vector into a leukemia cellline expressing p210, the cells did indeed revert to a normalphenotype. [See related article on GSEs on page 21.]