Pergamon Transfus. Sci.,Vol. 17, No. 1, pp. 29-34, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0955-3886{95)00055-0 0955-3886/96 $15.00 + O0 Gene Therapy with Physical Methods of Gene Transfer Kathryn E. Matthews* Armand Keating+ INTRODUCTION A wide variety of gene transfer method- ologies have been examined for the development of gene therapy and gene marking protocols. These include viral vectors, complexes of exogenous DNA with chemical compounds and several physical methods such as electropora- tion, bead transfection, particle bom- bardment and direct DNA transfer. When considering a target organ for the development of gene therapy/gene marking protocols, bone marrow cells, in particular multipotential stem cells, are particularly suitable as target cells since they can be extracted, manipulated and returned to the patient with relative ease. The pluripotent stem cell has the potential to remain throughout the life- span of the patient and to fully reconsti- tute haematopoiesis. It can be indirectly assessed for the presence of the trans- gene by analysing the cells comprising haematopoietic colonies using semi-solid methylcellulose assays for haematopoietic progenitors. ~ Since several genetic dis- eases such as the thalassemias, chronic granulomatous disease and immuno- deficiencies involving the lymphoid cells, affect one or more derivatives of haematopoietic stem cells, the ability to transfect stem cells may afford many "Institute of Medical Science, University of Toronto and Oncology Research, The Toronto Hospital Research Institute, Toronto, Canada. tAuthor for correspondence at: The Toronto Hospital, ml 2-036, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4. therapeutic opportunities? In addition, there is growing interest in protocols for the genetic marking of bone marrow cells, especially for determining the efficacy of purging and the origin of relapse after autologous bone marrow transplantation? ,4 Despite increasing numbers of clinical gene therapy/cell marking protocols using retrovirus (67/94 currently approved RAC proto- cols), this approach has drawbacks including the difficulty of introducing genetic material into the quiescent stcl:-~ cell population, achieving sustained high level expression of the transgene, '-,~ limitations regarding the size of the gene that can be transferred and the re- mote possibility of generating n~.w re- combinant viruses. ~' Other approaches include the use of adenovirus and adeno- virus-associated viruses (AAV). Adeno- virus transfection, while efficient, also has limitations that include lack of stable integration of the transferred DNA, restrictions on the size of the transgene, possible immunogcnicity7 and some safety concerns. '~ AAVs also have size limitations on the amount of exoge- nous DNA that it can carry `) and difficulty with sustained expression of the transgene in haematopoietic cells. "~ 29 ELECTROPORATION OR ELECTRIC- FIELD MEDIATED GENE TRANSFER Our laboratory has investigated an alternative strategy: electroporation, or