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

and were infected five times over a four day period beginning two days after
enrichment. Cells were counted using eosin exclusion and a hemacytometer.
Glucocerebrosidase enzymatic activity: Cells to be assayed were washed
twice in PBS, pelleted and stored at -80° C prior to analysis. Pellets were extracted
in cold 50 mM potassium phosphate buffer (pH 6.5) containing 2.5 mg/
ml Triton X-100. Ultrasonication (Branson Sonifier 450, power setting 8,5-10 seconds)
was used to disperse and lyse the cells, followed by 15 minute centrifugation
in a microfuge at 4° C to yield a clear supernatant for analysis. Enzyme activity
was determined by addition of one part lysate to three parts of synthetic
substrate (4-methyl-umbelliferyl glucopyranoside, Sigma Chemical Co, MO) at
10 mM in citric acid-sodium phosphate (0.12 M) buffer (pH 5.4) containing 2.5
mg/ml sodium taurocholate, 2 mg/ml Triton X-100, and 10 mg/ml bovine serum
albumin. The reaction was terminated after 30 minutes at 37° C by addition
of 0.17 M glycine-carbonate buffer (pH 10.4), and the fluorescence of the 4methylumbelliferone
product was measured with a fluorometer. Protein concentration
was determined using bicinchoninic acid (Pierce, Rockford, IL). Specific
GC enzymatic activity is expressed as nmoles per hour per mg of protein
(U/mg).
Southern hybridization: Southern hybridization was performed as previously
described (1), except that for DNA extraction the salt precipitation method
of Miller et al(6) was used without phenol extraction. Digestion of transduced
genomic DNA with SstI yields a 4.3 Kb diagnostic fragment which hybridizes to
the human GC cDNA probe.
RESULTS AND DISCUSSION
We have shown that significant glucocerebrosidase (GC) expression is
maintained in the tissues (bone marrow, spleen, thymus, and liver) of mice up to
seven months after transplantation of transduced bone marrow (1); the levels of
expression were consistent with high specific activity in the hematopoietic cells
in these tissues. Southern and enzyme analyses showed that virtually all the individual
spleen colonies from 2° animals transplanted with bone marrow from 4
to 7 month old 1° recipients contained and expressed the viral vector (Figure IA).
Now more than one year after the initial transduction, cytospins of peripheral
blood (PB) from 4/4 donor-positive 2° long-term recipients stain positive for the
human GC enzyme by immunohistochemistry (see Figure IB), indicating that
transduction initially occurred among some of the most primitive stem cells testable
in the murine model. Based upon these murine experiments, it appears that
certain requirements for clinical testing have been satisfied: long term expression
has been maintained in PB and in mature macrophages, very primitive stem
cells were initially transduced, and transduced cells have successfully reconstituted
hematopoiesis in many animals (37 2o recipients are now surviving) without
indication of pathology.
Infection of autologous bone marrow from Rhesus monkeys was contemplated
as the next step in preparation for human trials. Bone marrow from a
Rhesus monkey was infected by multiple exposures to high titer supernatant
from an amphotrophic producer line (y-crip) (Figure 2). It was evident from this
experiment that endogenous expression was higher in cells cultured in the presence
of M- and GM-CSF in comparison to IL-3, IL-6 and SCF, indicating that
240 SIXTH INTERNATIONAL SYMPOSIUM ON AUTOLOGOUS BONE MARROW TRANSPLANTATION