Plasmid insertion vectors that facilitate construction of herpes simplex virus gene delivery vectors

Gene, 154 (1995) 123-128 ©1995 Elsevier Science B.V. Alllhghts reserved. 0378-1119/95/$09.50

123

GENE 08670

Plasmid insertion vectors that facilitate construction of herpes simplex virus gene delivery vectors (Recombinant virus; gene therapy; immediate-early promoter; lacZ; interferon ~)

Jerry P. Weir a'b and Emelyn J. Dacquel b aDivision of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA; and 2The Department of Cellular Immunology, Walter Reed Army Institute of Research, and The Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD 20850, USA

Received by J.A. Engler: 21 July 1994; Revised/Accepted: 27 October 1994; Received at publishers: 2 December 1994

SUMMARY

Plasmid insertion vectors were designed for the expression of foreign genes in recombinant herpes simplex virus (HSV) vectors. One vecto:r, pGal9, was designed for the insertion of foreign genes with their own promoter; a second vector, pGall0, was designed for the insertion of coding sequences downstream from the HSV immediate-early 110K promoter. The 110K promoter directed efficient expression of foreign genes, particularly in replication-incompetent virus recombinants, as shown by the expression of the lacZ and IFN~ genes. These vectors should be useful for the characterization of various promoters for gene delivery, and for the efficient expression of foreign genes in a variety of cell types.

INTRODUCTION

Herpes simplex virus (HSV) vectors have been proposed for the delivery and expression of foreign genes in neurons and other p,~st-mitotic cells (reviewed in Breakefield and DeLuca, 1991). Although interest in HSV vectors has focused mostly on gene delivery to the neuron because of the natural ab!ility of the virus to establish a latent infection in that cell, it has become clear that HSV is a potential gene-delivery vector to other cell types. For example, replication-defective HSV vectors have been Correspondence to: Dr. J.P. Weir, Division of Viral Products, HFM-457, Center for Biologics Evaluatiort and Research, 1401 Rockville Pike, Rockville, MD 20852, USA. Tel. (1-301) 496-2696; Fax (1-301) 480-6124; e-mail: [email protected]

Abbreviations: 13Gal, 13-galactosidase;bp, base pair(s); HIV, human immunodeficiency virus; HSV, l~erpes simplex virus; ICP, infected cell protein; IFN, interferon; MCS, multiple cloning site; moi, multiplicity of infection; PAA, phosphonoacetic acid; PCR, polymerase chain reaction; pfu, plaque-forming u:ait(s); TK, thymidine kinase; tk, gene encoding TK. SSDI 0378-1119(94)00881-7

used for gene transfer to the liver for production of circulating Factor IX (Miyanohara et al., 1992), and to primary human monocytes for production of interferon (IFN~t) to inhibit human immunodeficiency virus (HIV) replication (Weir and Elkins, 1993). In each case, the target cell was normally non-proliferating and not susceptible to efficient retrovirus-mediated gene delivery. The two basic types of HSV vectors for gene delivery are plasmid-derived vectors, or amplicons (Spaete and Frenkel, 1982; Geller and Breakefield, 1988) and recombinant virus vectors. Although plasmid derived vectors require only manipulation of plasmid DNA and can contain multiple copies of the gene to be delivered, they require helper virus for propagation, and there is no evidence that they are maintained in the cell in a true latent form. In contrast, recombinant virus vectors, including replication-incompetent vectors, require no helper virus and can establish latency in neurons. The principal disadvantages are the potential cytotoxicity of the virus vector (Johnson et al., 1992), and the difficulty in manipulation of the HSV genome (Roizman and Jenkins, 1985).

124 The aim of the present work was to develop plasmid insertion vectors that would facilitate the construction of HSV vectors for foreign gene expression, particularly replication-incompetent vectors that might have potential as gene therapy vectors to non-dividing cells. In addition, we compare the expression of a foreign gene from one replication-incompetent virus vector under replicating and non-replicating conditions.

and contains a lacZ gene under the control of the Moloney leukemia virus long terminal repeat. In contrast to the expression of lacZ from the 110K promoter, lacZ expression in v8117-infected Vero cells was barely detectable above background. In E5 cells, 13Gal activity was highest at 48 h post-infection, but was less than 10% of that detected in vll0K-Gal-infected E5 cells (data not shown). These results indicate that the 110K promoter can direct efficient synthesis of foreign genes, particularly in replication-incompetent HSV vectors.

EXPERIMENTALAND DISCUSSION

(a) Expression of the lacZ gene from the immediate-early I l O K promoter In order to obtain vector-directed gene expression in the absence of viral replication, we used the promoter from the 110K immediate-early gene of HSV-1 to direct expression of a lacZ marker gene. This plasmid vector, pll0KP-Gal, was recombined into both replicationcompetent and incompetent viruses. The replicationcompetent HSV-1, vll0KP-Gal, was generated by transfection of pll0KP-Gal and HSV-I(F) DNA into Vero cells, followed by selection of progeny virus expressing the lacZ gene. Similarly, the replication-incompetent virus, vdl20/13-Gal, was made by transfection ofp110KPGal and HSV-1 d120 DNA into E5 cells. The virus d120 has a large deletion in the immediate-early 175K gene that codes for the essential infected cell protein 4 (ICP4), and can only be propagated on ICP4 complementing cell lines such as E5 (DeLuca et al., 1985; DeLuca and Schaffer, 1987). Progeny virus that expressed lacZ was purified by repeated plaque assay on E5 cells. Vero cells and E5 cells were infected with v110KP-Gal and vdl20/[3-Gal, and the expression of lacZ was determined by analysis of [3Gal activity in infected cells. Each virus produced higher levels of [3Gal activity in infected Veto cells than E5 cells (Fig. 2). Since vll0KP-Gal infected E5 cells and Vero cells equally well, as determined by plaque titration on both cell types (data not shown), the higher level of ]3Gal activity in Vero cells is probably due to the constitutive presence of ICP4 in E5 cells. During normal lytic infection, ICP4 represses expression from the 11OK gene (DeLuca and Schaffer, 1988). Infection with the defective virus vdl20/f3-Gal produced notably higher levels of [3Gal activity at each timepoint than infection with v110KP-Gal; approx, ninefold the level of [3Gal was produced in vd120/13-Gal vs. vll0KP-Gal-infected cells at 24 h post-infection. This difference is also probably due to the production of ICP4 in vll0KP-Gal-infected cells. For a comparison of the relative strength of lacZ expression, we also infected cells with the replication-incompetent HSV vector v8117 (Dobson et al., 1990). This vector is also ICP4-deficient

(b) Construction of the insertion vectors pGal9 and pGall0 An insertion vector for the delivery and expression of foreign genes other than lacZ in recombinant HSV would require some of the same features of pGal8, such as a MCS for insertion of desired genes, and tk sequences to direct homologous recombination. Disruption of the tk gene would facilitate isolation of the recombinant virus by allowing selection in the presence of acycloguanosine. Furthermore, insertion into the tk gene would be advantageous, at least in some circumstances, since tk-negative virus has been shown to establish latency in the trigeminal ganglia but not replicate or reactivate (Coen et al., 1989; Jacobson et al., 1993). In addition, a laeZ indicator would be advantageous, particularly when the other foreign gene does not provide a selectable marker. With these characteristics in mind, the pGal9 plasmid vector (Fig. 1B) was constructed from pGal8. An HSV-1 promoter, derived from the glycoprotein C (gC) encoding gene, was inserted upstream from the lacZ gene in pGal8 so that lacZ would be expressed during recombinant virus isolation. Foreign genes containing their own promoters can be inserted into the MCS of pGal9, and production of [3Gal from the co-transferred lacZ cassette can then be used to indicate recombinant virus. In order to express foreign genes using the l 1OK promoter, we constructed the plasmid insertion vector pGall0. This vector was derived from pGal9 by insertion of the promoter from the 110K immediate-early gene in the opposite orientation from the gC-lacZ gene (Fig. 1C). Two unique restriction sites remained downstream from the llOK promoter for the insertion of open reading frames.

(c) Gene expression from the replication-incompetent virus vdl20/IFNa We have previously described the use of a replicationincompetent HSV vector to deliver IFN~ to human monocytes as a means of inhibiting HIV replication in those cells (Weir and Elkins, 1993). This virus vector, vdl20/IFN~, was constructed by insertion of the coding sequences for human interferon a downstream from the

125

A

lacZ

Xbal Sa,~ Sphl

3.0 kb

Hincllll , \ X ~

BamHI

5' tk 1.3 kb

3' tk 1.8 kb

EcoRI

pGal8

2.6 kb

B

~'C Xloal promoter Sa~ Sphl

lacZ 3.0 kb

Hindlll ~

BamHI 5' tk 1,3 kb

/

~ ,

~

3'tk 1.8 kb

EcoRI

EcoRI

pGal9

2.6 kb

C

gC 11OK Xbal promoter ,,. promote

~"

lacZ

3.0 kb ~ ' ~ ,

HindIII . ~ . ~ . / ~ 3'tk 1.8 kb

5'tk 1.3 kb

'EcoRI

pGall 0

Bp.. EcoRI

11OK promoter in pGall0. To compare synthesis of IFN~ from this vector under replicating and non-replicating conditions, we infected Vero and E5 cells and examined the production of IFNu for 48 h (Fig. 3A). IFN0~ synthesis was greater in vdl20/IFN~-infected Vero cells than in E5 cells, reaching near maximal level between 24 and 48 h post-infection, and declining thereafter. Previously, we showed that in human monocytes IFN~ was synthesized for only three days after vdl20/IFN0t infection and that lacZ expression declined within five days of vdl20/[}-Gal infection. Thus, the 110K promoter may be primarily useful for short-term high-level gene expression in replication-incompetent vectors. As yet, there is no evidence to indicate whether the 110K promoter would be similarly expressed to high levels and then shut-off in vectorinfected neurons. In addition to the IFN~ gene, vdl20/IFN~ contains the lacZ gene under the control of the gC promoter, a well defined late HSV promoter. We further examined the expression of the lacZ gene in vdl20/IFN0~-infected cells (Fig. 3B). In E5 cells, lacZ was expressed from the gC promoter in the typical manner of an HSV late gene, first becoming detectable at 8 h post-infection and rapidly increasing between 12 and 24 h post-infection. Surprisingly, the lacZ gene was also expressed in Vero cells; approx, half as much 13Gal was detected at 24 h post-infection as in E5 cells. To determine whether lacZ expression from the gC promoter was influenced by the adjacent 110K promoter (Fig. 1C), we constructed a replication-incompetent virus that contained only the gC promoter and the lacZ gene inserted into the tk locus. Although lacZ was expressed as a late gene in infected E5 cells, there was no detectable expression in infected Vero cells (data not shown). This suggests that lacZ expression in vdl20/IFN0~-infected E5 cells is likely due to the juxtaposition of the 110K and gC promoters. (d) Replication of ICP4-deficient HSV vectors We investigated whether there was any replication of vdl20/IFN0~ or vdl20/[3-Gal in Vero cells, either because of the presence of replication-competent virus in stocks of virus prepared in E5 cells, or due to the ability of d120 and its derivatives to replicate at a low level in

2.6 kb Fig. 1. Structure of plasmid insertion vectors used to construct recombinant HSV vectors. (A) pGal8 has been described previously (Weir et al., 1990) for the analysis of promoter actvities. Promoter sequences from approx. - 600 (SacI site) to + 152 (NcoI site) of the 110K immediateearly gene were inserted into pGal8, using the HindIII and XbaI restriction sites, to give p110KP-Gal (B) pGal9 was derived from pGal8 by insertion of a gC promoter upstream from lacZ. The gC promoter from - l l 4 to +71 was derived from the plasmid pgCL5 (Weir and Narayanan, 1990) by PCR. TELe 5' end of the PCR fragment included an XbaI site and the 3' end was downstream from the BamHI site of

the lacZ gene, so that insertion of this fragment into pGal8 retained several unique restriction sites at the 5' end of the gC promoter. (C) pGall0 was derived from pGal9 by insertion of the HSV-1 llOK promoter sequences in the opposite orientation from the gC promoter. Routine procedures were similar to those described by Ausubel et aL (1987). Plasmid constructions were verified by sequencing before being used to construct recombinant viruses. All recombinant viruses were made by co-transfection of HSV-1 virion DNA and the appropriate plasmid DNA into Vero cells as previously described (Weir and Narayanan, 1988).

126

B

A

7O A m

E

>

7O =

Vero

60-

60-~

E5

50-

50-

40-

40-

= Vero ~

30~3

20-

20-

10-

1

0,~,

0

I

--

10

20

30

40

50

0

10

30

20

;0

50

Time after infection (h)

Time after infection (h)

Fig. 2. Infection of Vero and E5 cells with vIIOKP-Gal (A) or vd120/13-Gal (B). 106 Vero or E5 cells were infected with virus at moi 5, and at t h e indicated times were collected by scraping and centrifugation. They were then assayed for 13Gal essentially as described by Miller (1972), but adapted for 96-well plates. The A405 values were determined with a microplate reader and the activity of 13Gal in units/ml was determined by comparison with standards run in the same assay. Values reported are the mean of at least three separate experiments.

~"

B --

60] "-"~

X

F:5

5O

.>,

•~

~

40

~

30

o=

0

10

2"0

310

;0

Time after infection (h) Fig. 3. Production of IFN~ moi 5, and at the indicated using Madin-Darby bovine Interferon (Gxa01-901-535)

Vero

_-

50

0

10

20

30

40

50

Time after infection (h)

(A) and [3Gal (B) from vdl20/iFN~-infected Vero and E5 cells. 106 Vero or E5 cells were infected with vdl20/lFN~ at times the culture medium was examined for IFNcx bioactivity essentially as described previously (Coligan et al., 1991), kidney cells (ATCC CCL 22). Units of 1FNcx were determined by comparison with Reference Human Recombinant ~2 from the National Institute of Allergy and Infectious Diseases. I~Gal activity was determined as described in Fig. 2.

non-permissive cells. Plaque titration of all replicationincompetent virus stocks on both Vero and E5 cells failed to detect plaque-forming virus using Vero cells (Table I). Although we cannot completely rule out the presence of any replication-competent virus in these virus stocks, there would have been less than 32 pfu/ml, the limit of detection. In contrast, titers were greater than 1 x 108 pfu/ml in E5 cells. To investigate the possibility that replication-incompetent virus might replicate at a low level in Vero cells, cells were infected with either vdl20/[3-Gal or v d l 2 0 / I F N ~ at moi 5. After 2 h the inoculum was removed, the cells washed three times, and given fresh media. At 24 h post-infection, the cells and media were collected, the cells lysed by freeze-thawing, and the lysate

assayed on E5 cells. From 5 × 10 6 input virus, only 5 x 10 2 to 3 x 103 pfu/ml could be recovered after passage in Vero cells (Table I). In contrast, the same amount of input virus onto E5 cells resulted in > 3 x 10 v pfu/ml of recoverable virus (data not shown). It seems likely that the virus detectable after infection of Vero cells was due to residual input virus that was not removed by washing, since the same amount of virus was detected when the infection was done in the presence of phosphonoacetic acid (PAA) to inhibit viral replication (Table I). In control experiments, PAA lowered the amount of recoverable replication-competent HSV-1 by more than four logs (data not shown). These results suggest that ICP4-deficient HSV vectors do not replicate in the absence of ICP4, and that

127 TABLE I Growth of replication-incompetent virus Cell type for growth

Cell type for plaque assay~ Vero

E5

ICP4-producing cells but do not replicate in them, these vectors should be useful for gene delivery to a variety of cell types.

ACKNOWLEDGEMENTS

Virus s t o c k s vdl20/13-Gal vdl20/IFNct

E5 E5

< 32 <32

2 x 108 1 x 10s

Infecting virus b vd 120/l~-Gal + PAA vd120/IFNct + PAA

Vero Vero Vero Vero

n.d. n.d. n.d. n.d.

2x 3x 5x 5x

103 10a 10z 10z

a Virus was grown on E5 cells ~.fter infection at a multiplicity of 0.01 and harvested at 48 h post-infection. b Virus was passaged on 106 Vu~ro cells after infection at moi 5 and harvested at 24 h post-infection. c Numbers indicate pfu/ml (4 ml I. The limit of plaque detection was 32 pfu/ml, n.d., not done.

We thank Neal DeLuca (University of Pittsburgh, Pittsburgh, PA, USA) for E5 cells and virus d120, Anthony Dobson (University of California, Los Angeles, CA, USA) for virus 8117, and Ilan Sela (Hebrew University of Jerusalem, Rehovot, Israel) and Sidney Pestka (Robert Wood Johnson Medical School, Piscataway, N J, USA) for the I F N ~ gene. We thank Philip Krause and Michael Merchlinsky for helpful discussions.

REFERENCES

recombination between the ICP4 gene in E5 cells and the ICP4-deficeient virus d120 is extremely unlikely. Although there is no evidence to indicate that ICP4-deficient vector replicates in Vero cells, cellular toxicity was associated with vector infection, as described by others (Johnson et al., 1992). One day after vector infection of Vero cells at an moi of 5, there were approx. 40% as many viable cells as irt in mock-infected controls, as determined by trypan blue exclusion. This number decreased by half within Lwo days after vector infection (data not shown). Recent reports demonstrate that reduced expression of the remaining immediate-early genes significantly reduces the cellular toxicity of such replication-incompetent vectors (Johnson et al., 1994) (e) Conclusions (1) Plasmid insertion vectors have been designed to facilitate construction of :recombinant HSV vectors that express foreign genes. One vector, pGal9, designed for insertion of foreign genes with their own promoters, included a lacZ indicator gene to aid in detection of recombinant vectors and several convenient restriction sites for foreign gene insertion. A second vector, pGall0, also contained a lacZ indicator gene as well as the immediate-early 110K promoter and two convenient restriction sites for insertion of foreign gene coding sequences. Recombinant viruses can be selected in the presence of acycloguanosine for the expression of the co-transferred lacZ gene. (2) The immediate-early 11 OK promoter may be especially suited for high level short-term expression of foreign genes in replication-incompetent HSV vectors. (3) Since ICP4-deficienl: virus recombinants infect non-

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K.: Current Protocols in Molecular Biology, Greene and Wiley, New York, NY, 1987. Breakefield, X.O. and DeLuca, N.A.: Herpes simplex virus for gene delivery to neurons. New Biol. 3 (1991) 203-218. Coen, D.M., Kosz-Vnenchak, M., Jacobson, J.G., Leib, D.A., Bogard, C.L., Schaffer, P.A., Tyler, K.L. and Knipe, D.M.: Thymidine kinasenegative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate. Proc. Natl. Acad. Sci. USA 86 (1989) 4736-4740. Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M. and Strober, W.: Current Protocols in Immunology, Wiley, New York, NY, 1991. DeLuca, N.A. and Schaffer, P.A.: Activities of herpes simplex type 1 (HSV-1) ICP4 genes specifying nonsense peptides. Nucleic Acids Res. 15 (1987) 4491-4511. DeLuca, N.A. and Schaffer, P.A.: Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4. J. Virol. 62 (1988) 732 743. DeLuca, N.A., McCarthy, A.M. and Schaffer, P.A.: Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J. Virol. 56 (1985) 558-570. Dobson, A.T., Margolis, T.P., Sedarati, F., Stevens, J.G. and Feldman, L.T.: A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons. Neuron 5 (1990) 353-360. Geller, A.I. and Breakefield, X.O.: A defective HSV-1 vector expresses Escherichia coli 13-galactosidase in cultured periperal neurons. Science 241 (1988) 1667 1669. Jacobson, J.G., Ruffner, K.L., Kosz-Vnenchak, M., Hwang, C.B.C., Wobbe, K.K., Knipe, D.M. and Coen, D.M.: Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia. J. Virol. 67 (1993) 6903 6908. Johnson, P.A., Miyanohara, A., Levine, F., Cahill, T. and Friedmann, T.: Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1. J. Virol. 66 (1992) 2952-2965. Johnson, P.A., Wang, M.J. and Friedmann, T.: Improved cell survival by the reduction of immediate-early gene expression in replicationdefective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function. J. Virol. 68 (1994) 6347-6362.

128 Miller, J.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, pp. 352-355. Miyanohara, A., Johnson, P.A., Elam, R.L., Dai, Y., Witztum, J.L., Verma, I.M. and Friedmann, T.: Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX. New Biol. 4 (1992) 238-246. Roizman, B. and Jenkins, F.J.: Genetic engineering of novel genomes of large DNA viruses. Science 229 (1985) 1208 1214. Spaete, R.R. and Frenkel, N.: The herpes simplex virus amplicon: a new eukaryotic defective-virus cloning-amplifying vector. Cell 30 (1982) 295-304. Weir, J.P. and Elkins, K.L.: Replication-incompetent herpesvirus vector

delivery of an interferon a gene inhibits human immunodeficiency virus replication in human monocytes. Proc. Natl. Acad. Sci. USA 90 (1993) 9140-9144. Weir, J.P. and Narayanan, P.R.: The use of 13-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses. Nucleic Acids Res. 16 (1988) 10267-10282. Weir, J.P. and Narayanan, P.R.: Expression of the herpes simplex virus type 1 glycoprotein C gene requires sequences in the 5' non-coding region of the gene. J. Virol. 64 (1990) 445-449. Weir, J.P., Steffy, K.R. and Sethna, M.: An insertion vector for the analysis of gene expression during herpes simplex virus infection. Gene 89 (1990) 271-274.