Jul 17, 1987 - A transmissible retrovirusexpressing human hypoxanthine ... Clonal cell lines from the rat central nervous system. ... Cancer Inst. Monogr.
Vol. 8, No. 1
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1988, p. 457-460
0270-7306/88/010457-04$02.00/0 Copyright © 1988, American Society for Microbiology
Herpes Simplex Virus-Mediated Human Hypoxanthine-Guanine Phosphoribosyltransferase Gene Transfer into Neuronal Cells THOMAS D. PALELLA,1* LARRY J. SILVERMAN,' CHRISTOPHER T. SCHROLL,1 FRED L. HOMA,2 MYRON LEVINE,2 AND WILLIAM N. KELLEY"3 Departments of Internal Medicine,' Human Genetics,2 and Biological Chemistry,3 University of Michigan Medical School, Ann Arbor, Michigan 48109 Received 17 July 1987/Accepted 28 September 1987
The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B1034C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.
these reasons, HSV-1 was selected as a candidate for the transfer and expression of a human HPRT cDNA into cultured HPRT-deficient neuronal cells. This report details the isolation of HPRT- neuronal cells, the construction of chimeric HSV virions, the infection of these cells with the chimeric viruses, and the characterization of the expressed human HPRT. Infections were performed in an HPRT- derivative of rat neuroma cell line B103 (1, 9, 13, 18). B103 cells were rendered HPRT- by 'Co-irradiation-induced mutagenesis and selection in 14 ,ug of 6-thioguanine per ml (21). HPRTclones were isolated by limiting dilution. HPRT deficiency was confirmed by radioisotopic assay for HPRT (10) and by demonstration of nonviability in HAT medium (110 ,uM hypoxanthine, 2.3 ,uM aminopterin, 20 ,uM thymidine) (14). The HPRT- clone used in these studies was designated
The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to the mononucleotides IMP and GMP, respectively. The virtually complete deficiency of HPRT activity in humans results in Lesch-Nyhan syndrome, a devastating disease in which overproduction of uric acid and severe neurological dysfunction occur (12, 19). Despite the dramatic functional consequences of the enzyme defect, no anatomical or structural changes are noted in the central nervous systems of these patients. For this reason, as well as because of the lack of effective treatment, attention has focused on Lesch-Nyhan syndrome as one of the initial candidates for therapy by somatic-cell gene transfer (2). Retrovirus-derived vectors capable of expressing the human HPRT gene in cultured fibroblasts and lymphoblasts have been constructed by others (16). One of these constructs has been used to infect mouse bone marrow cells, which were then transplanted into lethally irradiated mice. These infected bone marrow cells repopulated the ablated hematopoietic cells of the recipient mice and transiently expressed human HPRT (15). However, it appears that correction of the enzyme deficiency in humans by this approach will be inadequate, since restoration of HPRT activity in the circulation by either exchange transfusion or bone marrow transplantation has no demonstrable effect on neurological function (7, 17). Hence, targeting the HPRT gene to the central nervous system appears to be essential for treatment of this disease. These observations indicate that it is likely that a vector must be capable of (i) being directed to neuronal cells, (ii) establishing a nondestructive relationship with a nondividing host cell, and (iii) expressing an inserted eucaryotic gene in a stable and efficient manner. Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that establishes latent infection in neuronal cells and, therefore, can establish a nonlytic relationship with the host cell (3, 5, 6). Furthermore, HSV-1 is capable of expressing foreign genes, including mammalian genes, inserted into its genome, in a variety of cell culture systems (20, 22). For *
B103-4C. The thymidine kinase (TK) gene is contained within a 3,560-base-pair BamHI fragment of the HSV-1 genome, BamHI Q (8). The viral TK promoter has been used to express foreign viral and eucaryotic genes in chimeric HSV constructs (20, 22). The strategy for constructing chimeric viruses containing the human HPRT cDNA (4) in an appropriate relationship to the HSV TK promoter is summarized in Fig. 1. Plasmid pXl contains the HSV BamHI Q fragment (map units 0.293 to 0.316) cloned into the BamHI site of pBR322 (8). A single BglII site is present in this plasmid, located within the 5'-transcribed noncoding region of the HSV TK gene. The human HPRT cDNA used in this construct is a 950-base-pair fragment including 100 base pairs of 5' untranslated sequence and 200 base pairs of 3' untranslated sequence (4). Synthetic BamHI linkers were ligated to the blunt-ended HPRT cDNA, and excess linkers were digested with BamHI. pXl was linearized with BglII and ligated to the BamHI-digested HPRT cDNA fragment. Escherichia coli DH1 was transfected with these recombinant plasmids. Plasmid DNA was digested with BamHI and subjected to Southern blot analysis with 32P-labeled HPRT cDNA as a probe to confirm the presence of HPRT sequences. These plasmids were then digested with HincIl to
Corresponding author. 457
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NOTES
MOL. CELL. BIOL.
HSV-TK
/
H~~~~
Bom Hi Q
Bq H
pXlI PBR 322
B H
HPRT cDNA (950 bp) 1. Blunt end 2. Bom HI linlm
I Bgl J G I
B H
ire
H B HG
B
B
H
-B H Bam HI
Bam HI H H
B
~~~~B
Bom HI
H
~
H
B
_J
Q'-40 Bom HI Q-87
1. Homologous recombination with intoct HSV genomic DNA 2. Ploque hybridizotion with 32P-HPRT cDNA. 3. Plaque purify HPRT virions.
HSV-HP87 HSV-HP40 FIG. 1. Strategy for construction of chimeric HSV containking human HPRT cDNA. Two viral recombinants, HSV-HP40 and HSV-HP87, were isolated. HSV-HP40 has HPRT cDNA in the correct orientation with respect to the TK promoter. HSV-HP87 1has HPRT cDNA in the opposite orientation. Drawings are not to sca:IIAe. Abbreviations: B, BamHI; G, BglII; H, HincIl; P, PstI; bp, b;ase
from the underside of the agarose, denatured, and hybridized to 32P-labeled HPRT cDNA as previously described (11). Plaques giving positive signals were picked, and virus was plaque purified three times. Two recombinant viruses were isolated: HSV-HP40, in which HPRT cDNA is in the sense orientation with respect to the TK promoter sequences, and HSV-HP87, in which HPRT cDNA is in the antisense orientation with respect to the TK promoter. Viral DNA extracted from cells infected with HSV-HP40 and HSV-HP87 was digested with BamHI and analyzed by Southern blotting to determine the site of insertion of the HPRT cDNA into the HSV genome as well as the number of copies incorporated. A single copy of HPRT cDNA was incorporated into each recombinant virus (Fig. 2). Furthermore, HPRT cDNA remained associated with the viral TK sequences as expected. B103-4C cells in confluent monolayers were infected with HSV-HP40 and HSV-HP87 at a multiplicity of infection of 4. Uninfected B103-4C cells and B103-4C cells infected with HSV-1 KOS were used as controls. Cells were harvested at 6, 20, and 30 h and lysed by repeated freezing and thawing. Cellular debris was removed by centrifugation, and the extracts were assayed for HPRT by determining conversion of [8-'4C]hypoxanthine to [8-14C]IMP as previously described (10). Results of these experiments are shown in Table 1. No HPRT activity was detectable in uninfected B103-4C cells or in HSV-1 KOS-infected B103-4C cells. In cells infected with HSV-HP40, HPRT activity was barely detectable at 6 h, but the levels rose significantly at 20 and 30 h. HPRT activity was also detected in HSV-HP87-infected cells at all three time points; compared with levels in HSV-HP40-infected cells, activity in HSV-HP87-infected cells was higher at 6 h and lower but significant at 20 and 30 h. To determine the origin (rat versus human) of the expressed HPRT activity in each experimental condition, cell extracts were electrophoresed through 6% polyacrylamide gels under nondenaturing conditions. Replicate gels were subjected to assay for HPRT activity in situ (Fig. 3A) and to immunoblot analysis (Fig. 3B) with polyclonal anti-human HPRT antiserum having no detectable cross-reactivity with rat HPRT (24). Human HPRT activity was detected by autoradiography only in normal human lymphoblast cell line GM 558 and in TABLE 1. Expression of HPRT activity in virus-infected
B103-4C cellsa
A
Postinfection HPRT activity (nmol/min per mg of protein)
pairs.
HPRT+ virus"
determine the sense or antisense orientation of the HP]RT cDNA with respect to the TK promoter (data not show n). Two plasmids, each of which contained a single copy of HPRT cDNA, which were designated pXl-HP40 and p)(1HP87, contained the cDNA insert in the sense and antiserise orientations, respectively. Chimeric virus was isolated f ollowing cotransfection of Vero cells by calcium phosph;ate precipitation with 2 ,ug of a BamHI digest of either p)(1HP40 or pXl-HP87, 0.5 ,ug of intact HSV-1 KOS DNA, a nd 7 ,ug of calf thymus DNA as a carrier (23). On day 3 posttransfection, virus was harvested by repeated free;zethaw cycles. Dilutions of this lysate were used to infiect confluent Vero cell monolayers which were overlaid with: 1% agarose and incubated at 37°C for 3 days, at which tiime plaques appeared. Plaques were blotted onto nitrocellulc)se
HSV-HP40 (sense orientation)
HSV-HP87 (antisense orientation)
at':
6h
20 h
30 h
0.07 0.04 0.04 0.04 0.29 0.16 0.20 0.15
9.60 4.38 1.36 5.01 1.30 0.46 0.97 0.87
ND 2.32 1.85 ND ND 0.30 0.75 ND
a HPRT activity in wild-type B103 cells was 2.80 + 0.35 nmol/min per mg (n = 8); in B103-4C cells it was
