Autologous Bone Marrow Transplantation - Blog Science Connections

Human ADA in Monkeys 713
4 were never fully reconstituted and the animals died before they could be
analyzed. In six of the eight remaining animals, human ADA activity could be
detected, ranging from less than 0.01% of endogenous monkey activity to
roughly 0.5% of endogenous activity. In four of the six human ADA-positive
animals, varying levels of NPT activity could also be detected. In only one
animal (Bill, no. 1) could vector DNA (representing less than 1/10 single-gene
dosage equivalents) be detected.
Such low levels of human ADA activity in these animals could represent
either near-endogenous levels of expression in a very few cells or very low
levels of expression in a large proportion of cells. Since the lower limit of
sensitivity of our Southern analyses is 1 /20 single-gene dosage equivalent,
and since in five of six human ADA-positive animals nonvector DNA could be
detected, it is possible that less than 5% of the hematopoietic cells in these
animals carry the SAX vector. In an effort to confirm that the low enzyme
levels observed were due to a small proportion of cells containing and
expressing vector-delivered genes, we performed in situ hybridization of
peripheral blood cells using a radioactively labeled probe specific for
transcripts from the bacterial neo R gene; results indicated that 28 of 3415
cells expressed the vector-derived gene. It must be noted, however, that
expression of the neo R
gene does not necessarily mean that the accompanying
human ADA gene is also expressed. Nonetheless, the sum of these data
suggest that probably less than 5% of bone marrow cells were infected and
that those infected were expressing human ADA at levels nearly equivalent to
those of the endogenous monkey ADA.
The levels of human ADA described here always are the peak levels
observed in any of the primates studied; in no animal were the peak levels
maintained. For example, in animal no. 7 (Kyle), the maximal level of 0.2% was
observed on day 104 posttransplant. Human ADA levels declined from this
maximum until no more activity could be detected by day 160 posttransplant.
Shortly thereafter (on day 181), a limiting dilution analysis (11) of peripheral T
cells was performed to determine if any cells could be detected that had
become resistant to the drug G418 as a result of the presence of the neo" gene.
In the absence of drug selection, 1 ¡1 of peripheral T cells could be cloned (Fig
3[line A]). After selection in 100 jug/m' G418 (a concentration at which
uninfected T cells were all killed), 1 /93 of the T cells from animal no. 7 could
still be cloned (Fig 3[ line B]). Therefore, approximately 8% of the clonable T
cells on day 181 posttransplant still carried expressing vector sequences. It
must be emphasized that such a limiting dilution analysis really only accounts
for a portion of all peripheral T cells. Therefore, the seemingly high
percentage of infected cells should not be interpreted as meaning that an
equally high number of total T cells carried functional vector. Rather, it may
only mean that at the time of assay, vector-containing T cells still were present
in the peripheral blood of the animal.