Autologous Bone Marrow Transplantation - Blog Science Connections

770 Human ADA in Monkeys
years in an active transplant program. The second was a more recently initiated
program at the National Institutes of Health using rhesus monkeys.
Our goal has been to develop a protocol that could be applied toward the
potential gene therapy of patients suffering from lethal genetic disease. The
initial disease targeted for study is the severe combined immunodeficiency
(SCID) caused by adenosine deaminase (ADA) deficiency. A number of
technical and medical reasons have been enumerated as to why ADA-SCID is
a likely candidate for gene therapy (6).
RESULTS
The vector used in the following experiments is one derived from the
Moloney murine leukemia virus and is based on the parental vector N2 (5). A
minigene consisting of a 1300-base pair (bp) sequence containing the fulllength
human adenosine deaminase-complementary DNA (ADA cDNA) (7)
regulated by a 400-bp fragment of the early promoter of the virus SV40 early
genes was inserted into the unique Xhol site of N2 (Fig 1) to create the vector
SAX. Cells producing the SAX vector at viral titres of 2 x 10 6 were isolated and
used to infect monkey bone marrow cells by one of the two protocols
described below.
The initial protocol for primates was a modification of one that we had
found to be capable of introducing the N2 vector into murine colony-forming
units from the spleen (CFC1-S) at an efficiency of over 85% (5). In this procedure,
bone marrow cells were aspirated from the long bones of an anesthetized
monkey and then nucleated cells were isolated by centrifugal sedimentation
through a Ficoll-Hypaque gradient. These nucleated cells were then exposed
SK P P E P P X EC SK
N2 | | rtssasssa^ )=| |
LTR 5> NEO R LTR
SK P P E P P P P E C S K
_ " II I I I I I II , U_
SAX I 1 IBMMWMMM Z = E ^ — — 3 = | I
LTR 5> NEO R SV40 hADA LTR
I • I i I i I i I i I • I
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
SCALE IN Kb
Figure 1. Retroviral vectors N2 and SAX. LTR, long terminal repeat; 5', retroviral 5' splice
donor site; P, packaging signal; A/eo", neomycin-resistant gene (cross-hatched area is
coding region); SV40, SV40 early promoter; hADA, human ADA cDNA. The symbols S, K,
P, E, X, and C refer to restriction endonuclease sites Sacl, Kpnl, Pstl, EcoRI, Xhol, and
Clal,
respectively.