JERUSALEM A limiting factor in gene therapy has been overcome: the problem of how to introduce genes into non-dividing bone marrow cells, the critical target for the cure of many heritable diseases. Monkeys provide the answer.
Research at the Hadassah University Hospital and the Hebrew University using the simian virus 40 (SV40) vector to introduce genes (instead of the retrovirus carriers commonly employed) will be presented at a meeting of the European Working Group on Gene Therapy, to be held in Jerusalem Nov. 21 through 24.
SV40 is not newly discovered. It was first found in batches of polio vaccines in the U.S. Millions of individuals of all ages were inadvertently infected with SV40 in the 1950s, as evidenced by the large numbers of Americans who test positive for antibodies to the virus. But SV40 has not been linked to any human disease in extensive epidemiological studies, after nearly half a century of follow-up.
¿We showed that with the simian SV40 pseudoviral vector, we could introduce the functional human MDR1 gene into cultured human bone marrow hematopoietic progenitor cells, with highly efficient gene transfer and with significant expression,¿ said Debbie Rund, hematologist at the Hadassah University Hospital and co-principal investigator with Ariella Oppenheim, professor of genetics in the department of experimental hematology in the Faculty of Medicine of the Hebrew University, of Jerusalem.
Other Cancer Trials Not Successful
The therapeutic MDR1 gene has been employed to try to reduce the toxicity caused by anticancer drugs in patients undergoing chemotherapy. MDR1 confers resistance of bone marrow cells to a number of naturally occurring, structurally unrelated types of chemotherapeutic agents, including anthracyclines, vinca alkaloids, paclitaxel and others.
MDR1 has been introduced to patients whose cancer does not involve the bone marrow, to spare the effects of chemotherapy on the hematopoietic system using retroviral vectors, but these clinical trials have been unsuccessful.
¿Our findings are very encouraging,¿ said Oppenheim. ¿In parallel to these gene-delivery studies showing the potential human therapeutic benefit, we are developing an in vitro packaging system for the SV40 vector in collaboration with Amos Oppenheim, professor of genetics in the department of molecular genetics of the Hebrew University of Jerusalem Faculty of Medicine. When established, it will make feasible the use of the SV40/MDR1 pseudovirion delivery system for chemotherapy protection in humans.¿
The in vitro packaging relies on the use of purified reagents: plasmid DNA prepared in bacteria and recombinant SV40 capsid proteins produced in insect cells, mixed together in the test tube under well-controlled, aseptic conditions.
The in vitro packaged pseudovirion meets the standards required for a drug prepared for human gene therapy, such as non-viral vectors (for example, naked DNA and liposomes), which are themselves very inefficient in gene delivery into many tissues, and are unable to deliver genes to hemopoietic cells; while, on the other hand, the packaged pseudovirion retains the highly efficient gene delivery of the simian virus.
Vector Easy To Handle, Transfer
¿These accomplishments have brought closer an actual cure for many heritable diseases including thalassemia, Gaucher¿s disease, severe combined immunodeficiency, and also various solid tumors,¿ said Rund, pointing out that the majority of gene transfer studies and clinical trials targeting hematopoietic stem cells use retroviral vectors, despite their numerous limitations.
Most retroviral vectors have difficulty penetrating into stem cells, despite expensive cytokine treatment. Other problems include sub-optimal engraftment of retrovirally transduced cells, as well as questionable safety issues.
¿It¿s puzzling,¿ Rund said. ¿Retroviral or adenoviral vectors for gene therapy still hold rank as the great hope for gene therapy, even though after many millions of dollars invested in [research and development], they are yet to produce success in clinical trials. In vitro, they seem to get into cells, but in actual patient trials they have been disappointing.¿
The SV40-based vector, in contrast, is easy to handle and efficiently gets into human hematopoietic cells, Rund said. The technique, she added, could have been introduced a decade ago if not for ¿conventional thinking that limited interest in the novel approach.¿
¿When the vector proves itself in human trials, the simian virus gene carrier would be the safest type of virus-facilitated gene transfer possible. Almost no viral sequences are included in the transferred DNA which would make them ideal for human use, for a variety of genetic disorders and certain cancers,¿ said Rund, referring to her team¿s research, published in the March 20 issue of Human Gene Therapy.
The joint Jerusalem group has applied for a patent covering the SV40 construct for gene therapy and one for the in vitro packaging, which they have established and demonstrated and are now making more efficient.
Oppenheim said efficiency, ¿to produce more pseudovirions per reaction, was increased 10-fold [recently]. Once we have reached adequate efficiency, SV40 will be ready for human use and clinical testing can proceed.¿