VI Autologous Bone Marrow Transplantation.pdf - Blog Science ...

TRANSDUCTION AND EXPRESSION OF THE HUMAN
GLUCOCEREBROSIDASE GENE IN THE LONG TERM MURINE MODEL,
RHESUS MONKEY BONE MARROW AND HUMAN CD34+ CELLS.
A. Bahnson, M. Nimgaonkar, S.S. Boggs, T. Ohashi, P.D. Robbins, K. Patrene,
J-F. Wei, Y. Fei, J. Li, E.D. Ball and J.A. Barranger.
Department of Human Genetics and Division of Hematology and Bone Marrow
Transplant, University of Pittsburgh.
Experiments conducted in mice indicate that retrovirus mediated gene
therapy may represent a viable treatment for Gaucher disease. Prior to initiation
of human trials, however, there should be clear indication that retroviral transduction
can lead to clinically relevant expression of the transferred
glucocerebrosidase (GC) gene in mature macrophages, since pathological storage
of glucosylceramide is restricted to these cells. As an indication that the
treatment can provide long term benefit, transduction of primitive marrow
repopulating stem cells should be obtained. In addition, the risks associated
with retroviral infection, i.e. proto-oncogene activation or insertional mutagenesis
leading to neoplastic transformation, must be low.
Recently we reported use of a retroviral vector, MFG-GC, in a murine model
of bone marrow transplantation.(l) This vector resulted in high levels of expression
in long-term reconstituted animals; such expression was commonly not observed
in previous studies.( 23
) Expression of human GC was demonstrated in
mature bone-marrow-derived macrophages cultured from primary (1°) recipients
4-7 months after transplant, and virtually 100% of the secondary (2°) spleen
colonies (produced from 1° bone marrow in 2° irradiated mice) contained transduced
vector sequences at this time. We report here additional preliminary findings
for 2° long-term reconstituted animals.
The murine model offers advantages not present for human studies, including
induction of stem cell cycling in the donor with 5-fluorouracil (5-FU) and the
option to use coculture of bone marrow cells with viral producer cells. The
former lacks effectiveness in primates (4), although other means of stem cell
stimulation may substitute. Coculture causes problems with FDA requirements
for human trials. We report here preliminary results obtained using filtered viral
supernatants for infection of Rhesus monkey bone marrow cells and of human
CD34 peripheral blood (PB) cells from G-CSF primed patients. The latter cells
may be appropriate targets for treatment of Gaucher patients.
MATERIALS AND METHODS
Viral vector and producer cells: The MFG retroviral vector was originally
constructed by Drs. Robbins and Guild in Richard C. Mulligan's laboratory
(Whitehead Institute, MA) and was modified in our laboratory by insertion of
the cDNA for human GC as described (1). The ecotropic producer, y-cre, was
used for transduction of murine cells. An amphotropic producer, y-crip, multi-
138 SIXTH INTERNATIONAL SYMPOSIUM ON AUTOLOGOUS BONE MARROW TRANSPLANTATION