Pntc. Nall. Acad. Sci. USA 
Vol. * *3. pp. 656J-6J67. September 1986 
Genetics 
Correction of adenosine deaminase deficiency in cultured human T 
and B cells by retrovirus-mediated gene transfer 
(retroviral vector/gene expression/gene therapy/lmmunodefkiency) 
Philip W. Kantoff**. Donald B. Kohn*, Hiroaki MitsuyaS, Donna Armentano*, Miri Sieberg^, 
James A. Zwiebel*. Martin A. Eglitis*. Jeanne R. McLachlin*. Dan A. Wiginton 11 , 
John J. Hutton 11 . Sheldon D. Horowitz**, Eli Gilboa 1 . R. Michael Blaese*. 
and W. French Anderson* 
‘Laboratory of Molecular Hematology. National Heart. Lung, and Blood Institute. National Institutes of Health. Bcthcsda. MD 70892; {Metabolism Branch 
and (Clinical Oncology Program. National Cancer Institute. National Institutes of Health. Bcthcsda. MD 70892; 'Department of Molecular Biology. 
Princeton University. Pnnceton. NJ 08544; 'Department of Pediatrics. Children's Hospital Medical Center. University of Cincinnati. 
Cincinnati. OH 45279; and "Department of Pediatrics. University of Wisconsin Hospitals. Madison. Wl 5)792 
Communii med by Rosette O. Beady. May 20. 1986 
ABSTRACT A retroviral vector called SAX, containing 
the cloned human cDNA for adenosine deaminase (ADA), has 
been constructed and used lo Introduce the ADA gene into 
cultured T- and B-lymphocyte lines derived from patients with 
ADA deficiency. DNA analysis showed that the SAX vector was 
inserted intact into the T and B celts at approximately one copy 
per ceil. The treated cells produced the characteristic isozymes 
of human ADA at a level similar to normal T and B lympho- 
cytes. It is known that ADA-deficient lymphocytes are unusu- 
ally sensitive to high levels of 2' -deoxyadenosine, and (his is the 
mechanism thought to underlie the selective lymphocytoloxk- 
ity associated with ADA deficiency in vivo. Expression of the 
introduced ADA gene was sufficient to reverse the hypersen- 
sitivity of these genetically deficient lymphocytes to 2'- 
deoxyadenosine toxicity. These results support the suggestion 
that retroviral vector gene-delivery systems show promise for 
application to human gene therapy. 
Retroviruses are able to transfer their genetic information at 
high efficiency into eukaryotic cells. These viruses can be 
genetically manipulated to replace their own genes with 
exogenous genes and thereby become vectors for gene 
insertion (1). Reports from several laboratories employing 
retroviral vectors (2-13) have demonstrated successful trans- 
fer and. in many cases, expression of exogenous genes in 
various hematopoietic cell types in vitro as well as in murine 
bone marrow stem cells. 
Successful human gene therapy will require efficient gene 
transfer as well as adequate expression of the delivered gene 
in appropriate target cells (1). An initial candidate for gene 
therapy is adenosine deaminase (ADA; adenosine aminohy- 
drolase, EC 3. 5. 4. 4) deficiency, a rare genetic disorder that 
underlies approximately one-quarter of all cases of severe 
combined immunodeficiency (SCID), a disease with pro- 
found defects of both cellular and humoral immunity (14. 15). 
ADA catalyzes the irreversible deamination of adenosine and 
deoxyadenosine to inosine and deoxyinosine. respectively 
(16). The molecular basis for the deficiency of ADA in most 
cases is the production of a caialytically defective enzyme 
molecule (17). The deficiency of ADA leads to selective 
toxicity affecting primarily T cells and. to a lesser and 
variable extent. B cells, with consequent severe immunologic 
dysfunction. This dysfunction is most likely caused by a toxic 
intracellular accumulation of deoxyadenosine and its metab- 
olites, particularly deoxyadenosine 5’-triphosphate (18. 19). 
The publication costs of (his arncle were defrayed in parr by page charge 
payment This article must therefore be hereby marked "ad\ rriisemenl" 
in accordance with 18 U.S.C. 117)4 solely to indicate this fact. 
We report here the use of a retroviral vector to study 
transfer and expression of the human ADA gene in ADA- 
deficient T- and B-lymphocytc lines. 
MATERIALS AND METHODS 
Generation of Viral Particle-Producing Cell Lines. The 
helper virus-free 3T3 packaging cell line PA-12 (5) was used 
to generate replication-defective viral particles containing the 
vector SAX. This cell line provides all the functions neces- 
sary for encapsidation of the vector RNA into a viral 
envelope capable of infecting a wide (i.e.. amphotropic) host 
range without producing replication-competent virus (5). 
PA-12 cells were transfected with SAX plasmid DNA by the 
method of Wigler el al. (20). Stably transformed clones were 
isolated by their resistance to the neomycin analogue G418 
(GIBCO). conferred by the function of the SAX neo R gene. 
These clones were then analyzed for viral production by 
serially diluting the supernatant from their cultures and 
adding it to 3T3 cells in the presence of Polybrene (8 /ig/ml; 
Aldrich). The medium was changed to selective medium (i.e., 
with G418 at 400 Mg/ml) 48 hr later, and G418-resistant 
colonies were scored after 10-12 days. More than 50% of the 
clones analyzed produced viral panicles containing SAX 
vector at titers greater than 5 x 10 3 colony-forming units 
(cfu)/ml. The clone used in these experiments had a viral titer 
of 2-5 x 10* cfu/ml. 
T- and B-Cell Lines. An ADA-deficient T-cell line (TJF-2) 
and two control normal T-cell lines (K7 and HM) were 
established by using the procedure of Mitsuya et al. (21) (see 
Results). T cells were maintained in RPM1 1640 medium 
supplemented with 2 mM L-glutamine. gentamicin (50 Mg/ 
ml), 10% heat-inactivated fetal bovine serum (FBS), and 10% 
(vol/vol) interleukin 2 (Cellular Bioproducts. Buffalo, NY) at 
37*C in 5% COj. Fluorescence-activated cell sorter (FACS) 
analysis of the TJF-2 cell line showed it to be uniformly 
positive forT3 (pan-T-cell marker). T9 (transferrin receptor). 
Til (sheep erythrocyte receptor). Tac (interleukin 2 recep- 
tor). and la (activated-T-cell marker). Approximately half the 
cells were T4-positive and half were T8-posiiive. The cells 
were uniformly negative for B1 (pan-B-cell marker) and for 
T6 and T10 (intrathymic lymphocyte markers). GM2756 and 
GM4258A. well-characterized ADA-deficient human B-cell 
lines, were obtained from the Human Genetic Mutant Cell 
Abbreviations: ADA. adenosine deaminase. HTLV-I. human T- 
lymphotrophic virus type I; LTR. long terminal repeat; SCID, severe 
combined immunodeficiency; SV40. simian virus 40; bp. base 
pain s). kb. kilobase(s). 
*To whom reprint requests should be addressed. 
6563 
Recombinant DNA Research, Volume 12 
[ 245 ] 
