Significantly Increased Expression of β-Glucuronidase in the Central Nervous System of Mucopolysaccharidosis Type VII Mice from the Latency-Associated Transcript Promoter in a Nonpathogenic Herpes Simplex Virus Type 1 Vector

METHOD

doi:10.1006/mthe.2000.0093, available online at http://www.idealibrary.com on IDEAL

Significantly Increased Expression of β-Glucuronidase in the Central Nervous System of Mucopolysaccharidosis Type VII Mice from the Latency-Associated Transcript Promoter in a Nonpathogenic Herpes Simplex Virus Type 1 Vector Jia Zhu,*,1 Wen Kang,* John H. Wolfe,†,‡ and Nigel W. Fraser*,2 *Department of Microbiology, School of Medicine, and †Department of Pathobiology and Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 ‡Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 Received for publication December 3, 1999, and accepted in revised form June 6, 2000

Herpes simplex virus (HSV) has the ability to establish life-long latent infections in postmitotic neurons and to remain transcriptionally active, continuously expressing latency-associated transcripts (LAT) while producing minimal disease. These properties have made HSV an excellent candidate for neuronal gene transfer. Previously, we have shown that in mucopolysaccharidosis type VII mice (MPS VII, β-glucuronidase deficiency) the LAT promoter is capable of expressing β-glucuronidase (GUSB) in the trigeminal ganglion and the brainstem after latency is established. However, the number of neurons expressing GUSB is much lower than the number expressing 2kb LAT following a wild-type virus infection. In this study, we have evaluated the effect of the position of the coding sequence relative to the LAT promoter on β-glucuronidase gene expression in the central nervous system (CNS). Non-neurovirulent (ICP-34.5-deleted HSV-1) vectors were used, allowing direct intracranial injection. Significantly more GUSB activity was detected in brains of MPS VII mice inoculated with a recombinant virus (HSV-LAT-GUSB-JS) in which the GUSB cDNA was inserted near the LAT promoter, compared to viruses where it was inserted farther downstream in either the LAT exon 1 or overlapping exon 1 and the 2-kb LAT intron. This vector produced more than 100 times the number of positive cells than the other constructs. During acute infection, the distribution of viral replication differed from the distribution of GUSB enzyme expression. Viral antigen was predominately present in cells around the site of injection in the caudate putamen and in ependymal cells lining the ventricles. In contrast, GUSB expression was present mainly in cells of the thalamus and hypothalamus, which did not exhibit viral antigen, suggesting that GUSB enzyme activity was expressed from latently but not acutely infected neuronal cells. This vector design should be useful for high-level expression of various genes in the CNS.

INTRODUCTION Genetic disorders affecting the central nervous system (CNS) can potentially be treated by gene transfer using vectors to deliver functional copies of defective genes into postmitotic neurons. The goal of such somatic gene therapy is to achieve stable and long-term expression of transferred genes. Among all the viral vectors used in neuronal cell gene delivery, the human neurotropic her-

1Present address: Department of Microbiology, Harvard Medical School, Building D, Room 608, 200 Longwood Avenue, Boston, MA 02115. 2Fax: (215) 898-3849. E-mail: [email protected].

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pes simplex virus (HSV) is distinguished by its ability to establish life-long latent infection in its host (1–3). After primary infection of skin or mucosal surfaces at the periphery, HSV-1 infects nerve endings at the site of infection and travels by retrograde axonal transport to the cell bodies in a ganglion of the peripheral nervous system. In the ganglion it forms latent infections in sensory neurons lasting for the lifetime of the individual (reviewed in 4 and 5). During latency, the viral genome is present as an episomal chromatin structure, from which no protein expression can be detected (1, 6). The lack of integration or expression of viral proteins during latency is a desirable quality for a viral vector. Viral gene expression during latent infection is strictly limited to MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD transcription from one gene located in the long repeat region of the genome, called the latency-associated transcript (LAT) region (7–9). The most abundant LAT is a 2kb non-polyadenylated RNA, which is an unusually stable intron that accumulates in the nuclei of latently infected neurons following splicing from the 8.3-kb primary transcript called minor (m) LAT (10–14). No LAT protein product has been detected, and the functions of LAT RNAs remain unclear. Although these transcripts are not required for the establishment of latent infection or reactivation (15–17), there are data supporting a role for LATs in the efficiency of these processes (15, 16, 18, 19). The LAT promoter and basal transcriptional regulatory elements are approximately 700 base pairs upstream of the 2-kb LAT intron and 29 bases upstream of the RNA start site (11, 20–23). Long-term expression of reporter genes from the LAT promoter has been achieved from its normal location in the repeat region of the viral genome. Constructs with the β-globin gene immediately downstream of the LAT promoter (11), a β-glucuronidase cDNA approximately 400 bp 3′ of the LAT TATA box (24), and the β-galactosidase gene at 0.8, 1.1, and 1.5 kb downstream of the LAT transcription start site (24) all showed prolonged, though low, levels of gene expression in the sensory neurons of latently infected mice. Expression of reporter genes from the LAT promoter when the expression unit is inserted in ectopic sites within the HSV genome can occur (25), but requires additional sequences downstream from the promoter for expression to be maintained (26, 27). These data have demonstrated the feasibility of expressing genes of interest in the nervous system using the LAT promoter. To test gene transfer into the CNS, we have used a well-characterized murine model of a human genetic disease, mucopolysaccharidosis (MPS) type VII (Sly disease) (28, 29). MPS VII is a lysosomal storage disease caused by a genetic deficiency of β-glucuronidase (GUSB). This lack of enzyme prevents glycosaminoglycan (GAG) degradation, resulting in a severe degenerative syndrome affecting a number of tissues including the brain (30). The MPS VII mouse contains a null mutation for the GUSB gene; thus, it expresses no GUSB activity, producing essentially the same disease as in humans. The MPS VII model provides an ideal experimental system to evaluate HSV-based vectors for somatic gene delivery to the nervous system because the transferred GUSB activity can be localized using an in situ reaction and assayed quantitatively (31). Previously we have shown that an HSV-based vector (17/LAT-RGUSB), in which a 2.4-kb rat GUSB cDNA was inserted approximately 400 bp downstream of the LAT TATA box, was able to express GUSB in both the trigeminal ganglia and brainstem cells of MPS VII mice up to 4 months postinfection (32). However, the number of GUSB-expressing neurons was very low in both the peripheral and central nervous systems (32, 33). The 17/LAT-RGUSB vector did not accumulate the 2-kb LAT because the cDNA insertion removed the splice donor MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

and the 5′ end of the 2-kb intron region. In situ hybridization experiments showed that the number of cells expressing LAT sequences was significantly reduced in animals infected with the vector virus compared to those infected with wild-type virus (32, 33). To evaluate whether disruption of the 2-kb LAT processing was affecting expression, we constructed vectors to maintain the integrity of the splice mechanism. The GUSB cDNA was inserted at two different sites in exon 1 and GUSB expression was compared with the original construct. Insertion of the cDNA near the transcriptional start site resulted in significantly higher levels of GUSB expression than with the other construct. Foreign gene expression from the vector appeared to be substantially greater than in previous studies using the LAT promoter, suggesting that this design may be useful for therapy as well as for expressing other experimental genes in the CNS.

MATERIALS

AND

METHODS

Cells and viruses. Vero cells were grown in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD), containing 5% calf serum, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The HSV-1 strain 17-based parental virus (1716) has a 759-bp deletion in both copies of the ICP-34.5 gene (34). Plasmids. All plasmids used in this study were constructed based on pGem7zf vector (Promega, Madison, WI). The target construct pXho-Sal (32) contains a 4.0-kb XhoI–SalI fragment from the BamHI E fragment of HSV-1 strain F, which represents nucleotides 9436 to 5464, based on the nucleotide sequence of HSV-1 strain 17 (35). The plasmid pBRB, which contains a 2.4-kb rat GUSB cDNA in place of the 0.9-kb BstEII–BstEII sequence downstream of the LAT promoter (32), was used to construct the recombinant virus GUSB-CD. The recombinant virus GUSB-JF has the 2.4-kb rat GUSB cDNA inserted at the second StyI site downstream of the LAT promoter. To construct pGUSB-JF, three subcloning steps were involved to create the correct GUSB insertion and to generate a plasmid with enough flanking sequence for homologous recombination. First, a 1.4-kb BsiWI–SrfI fragment digested from pXho-Sal was inserted into the SmaI site of pGem7zf by Klenow end fill-in and blunt-end ligation. The resulting plasmid, pJZ17, containing only the second StyI site, was linearized by StyI restriction enzyme and blunt-end ligated with a 2.4-kb EcoRI fragment of rat GUSB cDNA after Klenow end filling of both fragments. The resulting plasmid was called pJZ-47. Finally, the 3.4-kb RsrII–BsmI fragment from pJZ-47, which has a GUSB insertion in the right orientation, was cloned back into plasmid pXho-Sal by replacing the 1.0-kb RsrII–BsmI fragment. The 9.4-kb vector was designated pGUSB-JF and used for homologous recombination. The plasmid pGUSB-JS differs from pGUSB-JF in that the 2.4-kb GUSB gene was inserted at the first StyI site downstream of the LAT promoter. First, a 2.3-kb XhoI–BstEII fragment was cloned into the pGem7zf vector at the SmaI site by Klenow fill-in and blunt-end ligation. The resulting plasmid, pJZ-G8, contains only the first StyI site of the LAT sequence. Second, the 2.4-kb EcoRI fragment of GUSB cDNA was inserted into pJZ-G8 at the StyI site to generate plasmid pJZ-45. To obtain sufficient flanking HSV sequence for efficient recombination, a 3.0-kb NotI–RsrII fragment from pJZ-45 was inserted in pXho-Sal by substituting for the 0.6-kb NotI–RsrII sequence. The resulting 9.4-kb plasmid was designated pGUSB-JS. Construction of recombinant viruses. Recombinant viruses, GUSB-CD, GUSB-JF, and GUSB-JS, were constructed by homologous recombination between parental viral DNA and plasmid pBRB, pGUSB-JF, and pGUSB-JS DNA, respectively. One microgram of plasmid DNA, linearized by restriction enzyme XhoI, was cotransfected into Vero cells with 1 µg of parental

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METHOD HSV genomic DNA using Lipofectamine (Gibco-BRL) as previously described (36). After 3 to 5 days of culture, maximal CPE was observed and the transfected cells was harvested. The GUSB recombinant progeny were selected and purified by plaque-lift assay, as described previously (16). Briefly, Vero cells were infected with progeny virus at low multiplicity and overlaid with 1% agarose in medium. The viral plaques were then blotted onto Protran BA 85 nitrocellulose membrane (Shleicher & Shuell, Keene, NH) according to the manufacturer’s instruction. After Southern blot hybridization with radiolabeled GUSB DNA probe, positive plaques were picked and an additional three rounds of plaque purification were performed to ensure homogeneity of the recombinant virus stocks. Viral stocks were made at a multiplicity of infection of 0.01 PFU/cell, and titers were determined by plaque assays. Southern and Northern blot analyses. To confirm the genomic structure of recombinant viruses, 2 µg of viral DNA isolated by a modified Pignotti’s method (37) was digested with restriction enzyme BamHI, separated on 1% agarose gel, transferred to GeneScreen Plus membrane (NEN Research Products, Boston, MA), and hybridized with nick-translated [32P]dCTP-labeled GUSB probe (a 1.5-kb PstI fragment from the GUSB cDNA) and LAT probe (a 0.84-kb StyI–BstEII fragment from the LAT region). To analyze viral gene expression, total RNA was extracted from Vero cells infected with various recombinant viruses using TRIZOL reagent (Gibco-BRL). Polyadenylated RNA was purified from total RNA using the MicroPoly(A)Pure mRNA purification kit (Ambion, Austin, TX). Northern blot analysis was performed as described previously (7). Ten micrograms of total RNA or 1 µg of poly(A)+ RNA was treated with glyoxal, separated on a 1.2% agarose gel, vacuum blotted to GeneScreen Plus membrane (NEN Research Products), and hybridized with [32P]dCTP-labeled GUSB probe and LAT probe. Mouse infection and tissue preparation. The MPS VII mice were bred from the carrier strain B6.C-H-2bmI/ByBir-gusmps/+ (38). The mutant mouse can be identified using the simple PCR-based assay described previously (39). The MPS VII mice were inoculated intracranially with 2  105 PFU (2 µl) of recombinant virus into the left side of the caudate/putamen or hippocampus by a single site injection using a small animal stereotactic apparatus (Kopf Instruments, Tujunga, CA) as described previously (40). Each brain was removed by dissection, divided into forebrain, injection region–middle brain, and hindbrain. Each division was submersed in OCT embedding compound, immediately frozen in liquid nitrogen-cooled isopentane for 10 s, transferred to liquid nitrogen, and stored at −70C. The blocks were cut into 10-µm serial sections. Three serial tissue sections were used to perform immunohistochemistry staining (IHC), in situ hybridization (ISH), and in situ GUSB enzyme staining. Immunohistochemical procedures. Rabbit polyclonal antiserum to HSV1 (Dako, Carpinteria, CA) was used to detect replicating virus as described elsewhere (41). Antigen-expressing cells were detected by an indirect avidin–biotin immunoperoxidase method, using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with 3,3′-diaminobenzidine as the chromagen (42). In situ hybridization for detecting LAT and GUSB gene expression. ISH was performed as previously described (43, 44). Following proteinase K treatment, the tissue sections were hybridized with 35S-labeled DNA probe for 48 h at 50C. The slides were then washed, coated with NTB-2 nuclear track emulsion (Kodak, Rochester, NY), and stored at 4C. Following NTB-2 emulsion processing with developer and fixer (Kodak),

the sections were counterstained with eosin. The LAT and the GUSB probe were radiolabeled with [35S]dCTP by a nick-translation method and separated from unincorporated nucleotides by Sephadex G-50 quick spin columns (Boehringer Mannheim Corp., Indianapolis, IN). The specific activity of the probes was approximately 5  108 cpm/µg DNA. In situ GUSB staining. GUSB enzymatic activity in the frozen sections was detected and localized by using naphthol-AS-BI and β-D-glucuronide as substrates (31, 39). Active enzyme cleaves the substrates and in the presence of hexazonium pararosaniline a bright red precipitate is formed (29). Quantitative activity measurements were performed on sections as described previously (29, 45, 46). The values obtained for control tissues were lower than in previously studies due to the fixative used for performing the in situ hybridization protocol. All measurements in this study were performed on identically treated control and experimental tissues.

RESULTS Construction of Recombinant Viruses Previously we have shown that the LAT promoter is capable of driving GUSB gene expression in the trigeminal ganglia and brain stems of MPS VII mice during latency (32). However, the number of GUSB-expressing cells was significantly lower than the number expressing 2-kb LAT (32, 33). Since 17/LAT-RGUSB has a 0.9-kb BstEII–BstEII deletion, the impairment of gene expression may result from the interruption of exon 1 or the 2kb LAT intron. To test this hypothesis, two recombinant viruses (GUSB-JF and GUSB-JS) were constructed to restore the 2-kb LAT intron sequence by inserting the 2.4-kb rat GUSB cDNA into different sites (StyI) upstream of the 2-kb LAT splice donor signal (Fig. 1). No poly(A) addition signal was introduced with the GUSB cDNA; thus, the 2-kb LAT intron and other potential LAT products were not altered and the GUSB cDNA is embedded in the LAT exon. A non-neurovirulent strain of HSV (strain 1716) was chosen as the parental virus because it is deleted in both copies of the ICP-34.5 gene, which allows direct intracranial inoculation into the MPS VII mouse brain (47). The only difference between the GUSB-JF and the GUSB-JS viruses was the 371-base-pair difference in distance from the LAT promoter to the GUSB cDNA sequences. Since the LAT sequence is diploid in the HSV genome, the recombinant viruses have two copies of the GUSB cDNA inserted at sites 7116 and 119249 of the HSV viral genome for GUSB-JF and at positions 7487 and 118878 on the HSV genome for GUSB-JF (35). The genomic structure was verified by Southern blot analysis using LAT and GUSB probes (Fig. 2).

FIG. 1. Map of HSV-1 and recombinant viruses. (A) The prototype HSV-1 genome in which the unique long and short regions are represented as lines (UL and Us) and long and short repeat regions are represented as open boxes (TRL, IRL, IRs, TRs). (B) A detailed map of BamHI B, SP, and Y fragments corresponding to the LAT gene showing the location of various restriction sites and transcripts in this region. (C) An enlarged LAT region from PstI (118659) to MluI (121636) including the LAT promoter, exon 1, 2-kb intron, and part of exon 2. 1716, the parental virus, has a 759-bp deletion in both copies of the ICP-34.5 gene. In recombinant virus GUSB-CD, the 0.9-kb BstEII fragment was replaced by the 2.4-kb rat GUSB cDNA disrupting the 5′ end of the 2-kb LAT intron. While in recombinant virus GUSB-JF and GUSB-JS, the 2.4-kb GUSB cDNA was inserted into sites upstream of the 2-kb LAT, at positions 119249 and 118878 in exon 1, respectively. (D) Map of LAT exon 1 showing the positions of the StyI insertion sites and the ATGs between them.

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MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD

MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

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METHOD

FIG. 2. DNA analysis of GUSB-recombinant viruses. (A) Diagram showing the expected structure of the BamHI E and BamHI B regions of the parental virus and GUSB-recombinant viruses. The major restriction enzymes located in the BamHI B, BamHI E, and GUSB cDNA as well as their corresponding sizes are marked. B, BamHI; S, StyI; BE, BstEII; P, PstI. The expected sizes of fragments are (1) for parental virus, 8.9- and 10.1-kb fragments which hybridize only with the LAT probe; (2) for GUSB-CD, 7.4 and 6.2 kb which only hybridize with the GUSB probe; (3) for GUSB-JF, 4.2 kb which hybridizes with the LAT probe and 7.4 and 6.2 kb which hybridize with the GUSB probe; and (4) for GUSB-JS, 4.6 kb which hybridizes with LAT probe and 7.1 and 5.9 kb which hybridize with the GUSB probe. (B) Two micrograms of viral DNA isolated from Vero cells infected with parental virus (lane 1), GUSB-CD (lane 2), GUSB-JF (lane 3), and GUSB-JS (lane 4) was digested with restriction enzyme BamHI prior to separation through 1% agarose gel by electrophoresis and probed either with a 0.84-kb StyI–BstEII fragment of the LAT region or with a 1.5-kb PstI fragment from GUSB cDNA.

LAT and GUSB RNA Are Expressed in Vero Cells Infected with the Recombinant Viruses To examine expression of the 2-kb LAT intron and GUSB mRNA in recombinant viruses, RNA was extracted from infected Vero cells at 12 h postinfection and probed on Northern blots. The 0.84-kb StyI–BstEII probe was specific for the 2-kb LAT sequence (Fig. 3). This probe detected 2-kb LAT in cells infected with the parental virus and the recombinant viruses GUSB-JF and GUSB-JS, but did not detect any transcripts in the cells infected with the GUSB-CD virus. The 2-kb LAT expressions from

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the recombinant viruses were 5- to 10-fold lower than that from the parental virus (Fig. 3). In addition to the 2kb LAT, there was a 2.3-kb transcript detected in GUSBJF-infected cells and a 2.7-kb transcript in GUSB-JSinfected cells. This may be due to a cryptic splice donor signal at the 3′ end of the GUSB cDNA sequence which is utilized in the processing of the LATs from these viruses (48). The different transcript sizes between JF and JS reflect the 371-bp difference in the site of GUSB insertion. To detect GUSB expression, poly(A)+ RNA was selected and subjected to gel electrophoresis and Northern MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD blotting followed by hybridization with a 1.5-kb GUSBspecific PstI fragment probe. Transcripts of about 9.1-kb for GUSB-CD and 10 kb for GUSB-JF and GUSB-JS were detected (Fig. 3). Since no exogenous poly(A) addition sequences were added to the LAT locus, these transcripts are consistent with use of the poly(A) signal of the mLAT primary transcript (49). A smaller poly(A)+ GUSB-specific transcript of approximately 7 kb was detected in all three recombinant viruses (Fig. 3). These RNAs may be transcribed from other promoters or from the LAT promoter but spliced differently. Although potentially interesting, the detailed mapping of these transcripts was beyond the scope of this study.

FIG. 3. Northern blot of GUSB-recombinant virus RNA. Ten micrograms of total RNA or 1 µg of polyadenylated RNA was extracted from Vero cells infected with parental virus (lane 1), GUSB-CD (lane 2), GUSB-JF (lane 3), and GUSB-JS (lane 4) at 12 h postinfection and hybridized with the LAT probe (0.84-kb StyI–BstEII fragment) or the GUSB probe (1.5-kb PstI fragment), respectively.

GUSB Expression in MPS VII Mouse Brains 4 Days after Intracranial Injection with GUSB-Recombinant Viruses To evaluate GUSB gene expression in the CNS, the recombinant viruses were directly injected into the caudate/putamen of adult MPS VII mouse brains. Sections of brain were assayed using a histochemical reaction for

TABLE 1 -Glucuronidase Activity in MPS VII Mouse Brains Infected with Parental or GUSB-Recombinant Viruses Mouse

Days of infection

Number of brain sections

Total GUSB-positive cells

PBS

3974

Acute 4

120

0

0

1716

3931

Acute 4

112

0

0

GUSB-CD

3472

Acute 4

108

3

0.03 ± 0.02

3584

Acute 4

96

2

0.02 ± 0.02

3882

Acute 4

90

2

0.02 ± 0.02

3962

Acute 4

60

1

0.02 ± 0.02

3497

Latent 28

84

2

0.02 ± 0.02

3499

Latent 28

102

2

0.02 ± 0.01

3566

Latent 28

96

1

0.01 ± 0.01

5015

Acute 4

210

59

0.3 ± 0.2

5135

Acute 4

150

53

0.4 ± 0.3

6302

Acute 4

120

22

0.2 ± 0.1

6304

Latent 28

104

11

0.1 ± 0.0

6293

Latent 28

104

8

0.1 ± 0.0

6147

Acute 4

48

696

14.5 ± 4.1

6153

Acute 4

152

7,744

51.4 ± 10.0

6163

Acute 4

152

3,328

21.9 ± 3.1

6165

Acute 4

104

5,064

48.7 ± 10.2

6154

Latent 14

104

9,280

89.2 ± 14.5

6197

Latent 14

144

12,824

89.1 ± 15.9

6199

Latent 28

112

2,456

21.9 ± 3.5

6170

Latent 28

128

12,160

95.0 ± 15.4

6167

Latent 43

144

4,630

32.2 ± 8.2

Virus

GUSB-JF

GUSB-JS

GUSB+/section (mean  SE)

Note. MPS VII mice were ic inoculated with various viruses as described under Material sand Methods. At the indicated time after infection, the animals were sacrificed and their brains were sectioned and assayed for GUSB enzyme activity. MPS VII mice were also mock infected with PBS or viral infected with parental virus 1716 as controls.

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METHOD

FIG. 4. Acute viral infection, RNA expression, and GUSB enzymatic activity in representative fields of MPS VII mouse brains intracranially inoculated with recombinant viruses. (A) MPS VII mice were intracranially injected with GUSB-CD (panels 1–4), GUSB-JF (panels 5–8), and GUSB-JS (panels 9–12). After 4 days postinoculation, brain sections were analyzed by immunohistochemistry (IHC), in situ hybridization (ISH), and GUSB staining to detect viral infection, LAT, and GUSB RNA expression and GUSB enzyme activity. Panels 1, 5, and 9: Brown staining shows viral infection at ependymal cells lining the lateral ventricle (panel 1), third ventricles (panels 5 and 9), and a few cells in the surrounding area. Panels 2, 6, and 10: Bright-field view of ISH showing GUSB RNA expression at the corresponding region on the adjacent section. Panels 3, 7, and 11: Dark-field view of ISH showing the LAT RNA expression at the corresponding region. Panels 4, 8, and 12, GUSB staining reaction shows no GUSB enzyme activity detected at ependymal cells along the ventricles on the adjacent sections in all three virus-infected mouse brains (panels 4, 8, and 12), but positive cells (red histological staining) were detected at the hypothalamus area distal to the third ventricle in GUSB-JS-infected mouse brains (panel 12). Magnification: 4 (panels 1–12). (B) MPS VII mice were ic inoculated with recombinant virus GUSB-JS. After 4 days of infection, brains were dissected and assayed by IHC using HSV-1 polyclonal antibody and GUSB enzyme staining at an adjacent section. IHC indicating viral infection at an inoculation site at caudate/putamen (panel 1) and ependymal cells along the dorsal third ventricle (panel 3) as shown by brown dots. GUSB enzyme activities, marked by red dots, were detected at the inoculation site in the section (panel 2) and the habenular nuclei under the dorsal third ventricle but not at the ependymal cells lining the ventricle (panel 4). Magnification: 4 (panels 1–4).

GUSB enzymatic activity, which can detect single positive cells (39) (e.g., see Fig. 5). Four days after infection (the acute stage) with GUSB-CD, the numbers of GUSBexpressing cells were very low, on average 2 positive cells per 100 brain sections (Table 1). The number of positive cells was comparable to that seen previously in the trigeminal ganglia and brainstems when mice were infected with HSV 17/LAT-RGUSB by corneal inoculation

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(32). Mice infected with recombinant virus GUSB-JF, in which the complete 2-kb LAT intron sequence was present, had an average of 45 GUSB-positive cells per 100 brain sections (Table 1). The GUSB-positive cells were found mainly in the thalamus (data not shown). The number of GUSB-positive cells was 14-fold higher in GUSB-JF-infected brain tissue than that infected with GUSB-CD virus (Table 1), suggesting that the presence of MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD

FIG. 5. Comparison of expression of GUSB enzyme activity during acute and latent infection. In situ GUSB staining showing the GUSB activities in the CNS of MPS VII mice inoculated into the caudate/putamen region of the brain with virus GUSB-JS (panels A1–A4). After 4 days of infection, GUSB-positive cells (red histological staining) were detected at the thalamus (1, both sides of the habenular nuclei under the dorsal third ventricle; 2, thalamic nuclei group) and the hypothalamus area (3, hypothalamic nuclei; 4, preoptic nuclei). Detection and distribution of GUSB enzyme activity in mouse brain latently infected with virus GUSBJS. Panels B1 to B3 representing GUSB enzyme activity in mouse #6197 brain section at positions approximately −1.70 mm (panel B1), −2.06 mm (panel B2), and −2.54 mm (panel B3) to the Bregma line according to the mouse brain atlas (68). The GUSB expression was widespread in the thalamus and hypothalamus. The GUSB expression level and distribution pattern were found to be similar between mice #6154, #6197, and #6170. The blue background in panels 1–4 were due to a blue filter used to emphasize the red enzyme activity. No filter was used for panels B1–B3. Magnification: 4 (panels 1–4), 1 (panels B1–B3).

the 2-kb LAT intron (or the 0.9-kb BstEII–BstEII LAT sequence) may facilitate expression of GUSB enzyme activity from the LAT-GUSB gene. In contrast, injection of recombinant virus GUSB-JS resulted in many more GUSB-positive cells (Table 1) than injection of GUSB-CD or GUSB-JF viruses. The average number of positive cells was 34.1 per brain section, which was more than 100 times higher than with GUSBJF and 1700 times higher than with GUSB-CD. The variation of GUSB expression from mouse to mouse was about threefold. The abundant numbers of GUSB-positive cells in the GUSB-JS-infected brains were found predominantly in the thalamus and hypothalamus (Fig. 5A, panels 1–4). Taken together, the difference in GUSB expression among the three recombinant viruses suggests that the 2-kb LAT intron and its upstream sequence (exon 1) are involved in the regulation of LAT-GUSB chimeric gene expression.

GUSB Activity in GUSB-CD and GUSB-JF VirusInfected Mouse Brain Does Not Correlate with GUSB RNA Expression To understand the mechanism of differential expression between GUSB-CD, GUSB-JF, and GUSB-JS vectors, MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

serial sections were used to compare of the distribution of viral antigens, LAT RNA, and GUSB enzymatic activity (Fig. 4). Cells positive for HSV-1 antigens, indicative of replicating virus, were detected predominantly around the injection site and in the ependymal cells lining the ventricles, with few positive cells at distant sites. These findings are consistent with previous studies (50–52) and were observed for all three GUSB-recombinant viruses (Fig. 4A, panels 1, 5, and 9). To determine whether the lack of GUSB enzymatic activities from GUSB-CD- or GUSB-JF-infected brain tissue resulted from a lack of GUSB RNA expression, in situ hybridization was performed using probes to both the 2kb LAT and the GUSB sequences. The MPS VII mouse brain has no detectable hybridization with GUSB due to the mutation (29, 33, 47). Cells positive for GUSB RNA were abundant at the site of injection and in ependymal cells lining the ventricles with both GUSB-CD (Fig. 4A, panels 2 and 3) and GUSB-JF (Fig. 4A, panels 6 and 7) vectors. Some positive cells were also seen in the motor cortex, thalamus, and hypothalamus (not shown). The GUSB RNA expression colocalized with expression of the LAT RNA, and the expression pattern matched well with the pattern of viral antigens. However, for viruses GUSBCD and GUSB-JF, no GUSB activity was detected on the

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METHOD mus, compared to 26.1 GUSB-positive cells in the habenular nuclei of the thalamus and 33.4 GUSB-positive cells in the hypothalamus (Fig. 6).

Distribution of GUSB Expression in the CNS of MPS VII Mice Latently Infected with Recombinant Virus GUSB-JS

FIG. 6. The comparison of GUSB expression and viral replication in the thalamus (habenular nuclei) and hypothalamus of brains acutely infected with recombinant GUSB-JS virus. MPS VII mice were ic inoculated with virus into the left side of the caudate/putamen. After 4 days of infection, brains were dissected and assayed by in situ GUSB enzyme staining and IHC using HSV-1 polyclonal antibody. Adjacent brain sections were counted for the number of GUSB-positive and HSV-1 antigen-positive cells at the habenular nuclei of the thalamus and the hypothalamus. The GUSB-positive cells per brain section (means ± SE) were 26.1 ± 3.0 at the habenular nuclei of the thalamus and 33.4 ± 2.0 at the hypothalamus. The numbers of HSV-1 antigenpositive cells at the habenular nuclei and the hypothalamus area per each brain section (means ± SE) were 1.0 ± 0.2 and 2.5 ± 0.3, respectively.

adjacent sections using the in situ histochemical reaction assay (Fig. 4A, panels 4 and 8).

The Distribution of GUSB Enzymatic Activities Was Different from the Distribution of HSV-1 Replication in Virus GUSB-JS-Infected Mouse Brains In contrast to the GUSB-CD and GUSB-JF vectors, abundant GUSB enzyme activity was detected in MPS VII mouse brains 4 days after inoculation with the GUSB-JS virus (Table 1). However, the distribution of GUSB enzyme activity was different from that of virus replication, as detected by immunohistochemical staining for viral proteins. As in the CD and JF vectors, viral antigens and RNA were detected predominantly at the site of injection and in ependymal cells lining the ventricles (Fig. 4A, panels 9–11), while almost no GUSB activity was detected in these areas. In contrast, abundant GUSB-positive cells were detected deep in the posterior hypothalamic area distal to the third ventricle (Fig. 4A, panel 12). At the injection site, viral antigen-positive cells were present in large numbers (Fig. 4B, panel 1), but GUSB enzymatic activity was minimal (Fig. 4B, panel 2). Furthermore, the GUSB-JS vector expressed abundant GUSB activity in the thalamus and hypothalamus (Fig. 4B, panel 4). The number of viral antigen-expressing cells was significantly lower than the number of GUSBexpressing cells (Fig. 4B, panel 3). On average, there was only 1 virus-positive cell in the habenular nuclei of the thalamus and 2.5 virus-positive cells in the hypothala-

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If the GUSB enzymatic activity detected at 4 days postinfection with virus GUSB-JS was indeed from latently infected neuronal cells, the same distribution of GUSB expression would be expected at later latent time points. To test this, MPS VII mice inoculated with GUSB-JS were sacrificed after latency was established, and the brain tissues were examined for viral infection and GUSB expression. Immunohistochemical staining using HSV-1 polyclonal antibody revealed no positive sections (data not shown), consistent with previous data showing that the ICP-34.5 mutant virus is efficiently cleared from the nervous system by day 7 (50–52). In contrast, abundant GUSB-positive cells were found (Fig. 5B). The number of GUSB-positive cells ranged from 21.9 to 95.0 per brain section (Table 1). GUSB expression from the GUSB-JS vector was expressed in a significantly greater number of cells (Table 1) than the GUSB-CD (P < 0.001) and GUSBJF viruses (P < 0.01) (ANOVA). Although the numbers of cells positive for GUSB during latency (average of 65 per brain section) were higher than that during acute infection (average of 34 per brain section), the difference was not statistically significant. Cells staining positive for GUSB were detected throughout the thalamus and hypothalamus, but only a very few positives were seen in the cortex, hippocampus, or ependymal cells lining ventricles (Fig. 5B, panels 1–3). The distribution pattern of GUSB expression during latent infection was similar to that during acute infection (Fig. 5A, panels 1–4). This pattern was different from that of viral antigen-positive cells during acute infection (Fig. 4, panel 9), again indicating that the GUSB activity was expressed from latently infected neuronal cells at 4 days postinfection but not in cells in which the virus was replicating. In Fig. 5B, panels 1–3 represent three brain sections of mouse #6197 at positions approximately − 1.70 mm (U), −2.06 mm (V), and −2.54 mm (W) posterior of bregma. Substantial numbers of positive cells were found on the contralateral side, and distal from the injection site, indicating that the spreading probably occurred through axonal transport.

Recombinant Virus GUSB-JS Produces Measurable Levels of GUSB Enzymatic Activity in Latently Infected MPS VII Brains Previous studies on HSV vectors have resulted in too few GUSB-positive cells to measure enzymatic activity above background (32, 47). The amount of GUSB staining seen in the GUSB-JS vector virus-infected brains suggested that activity could be present in measurable MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD amounts. Sections from the three brains with the highest number of positive cells, mice 6154, 6197, and 6170 (Table 1), were selected for assay. The distribution patterns of GUSB-positive cells were similar in these three animals (not shown). The GUSB enzymatic activities were measured on lysates of sister sections of those stained in the histochemical localization assays and were well above background (Table 2). The region of highest activity (slide D) corresponded to the sections with the highest number of positively staining cells (Fig. 5B, panel 3). These levels suggest that the GUSB-JS vector may be able to correct storage in future therapeutic trials.

DISCUSSION A major problem for gene transfer vector design is the achievement of long-term, stable, and high-level expression. Many vectors show high initial levels of expression but have declining levels over time, due to vector inactivation or loss of transduced cells. HSV-1 has many qualities that lend it to gene transfer to the nervous system. In the formation of a latent infection in neuronal cells, the genome exists in the nucleus of the infected cell in a nonintegrated chromatin form and lasts for the lifetime of the infected animal (1, 3, 53). Thus, HSV-based vectors overcome the problem of integration into the host genome and provide a potentially life-long platform for gene expression. Because the LAT region is the only viral sequence expressed at high levels after latency is established, the LAT promoter has been a candidate for long-term expression in the nervous system. However, the amount of gene expression from this promoter has generally been disappointingly low (32). Some studies suggest that the LAT core promoter, including the TATA box and about 800 bp of upstream sequence, may not be sufficient to drive long-term gene expression during latency but may

require additional DNA sequences downstream from the LAT transcriptional start site (11, 24–27, 54). Other studies have found that these downstream sequences may also affect expression from the LAT promoter either directly or through influencing the efficiency of latent cell formation (19, 55, 56). In the MPS VII model, we previously found that a small number of cells maintained long-term expression in the CNS after peripheral inoculation of a HSV-1 vector in which the cDNA disrupted the boundary of the LAT exon 1 2-kb intron region (32). However, the vector was less efficient than the parental strain at establishing a transcriptionally active state during latency in neurons, suggesting that interruptions in the 2-kb LAT intron interfere with efficient LAT promoter-driven gene expression (33). Thus, we constructed vectors with the GUSB cDNA inserted at two different positions within exon 1 of the LAT gene. The 2-kb intron was left intact and used as a marker for latently infected cells. These viruses were compared to the previously studied construct (32, 33) except that the viruses constructed for this study used the HSV-1 1716 virus which has a deletion in the ICP34.5 gene (34), a nonpathogenic variant which permits direct intracranial inoculation into GUSB-deficient mice (47). To minimize disruption to the LAT gene, we inserted the GUSB cDNA without attaching an exogenous poly(A) addition signal. By using the natural poly(A) signal of the full-length LAT transcript (the 8.3-kb minor LAT), transcription and splicing from the LAT locus should be minimally altered (57, 58). The GUSB RNA expressed from all three GUSB-recombinant viruses were in the mLAT form. Using a GUSB probe, a polyadenylated transcript of about 9.1 kb was detected in Vero cells infected with recombinant GUSBCD virus and about 10 kb in cells infected with viruses GUSB-JF and GUSB-JS (Fig. 3B). The sizes of the large transcripts were consistent with transcription being initi-

TABLE 2 GUSB Enzymatic Activity in MPS VII Mouse Brains Mouse

Genotype

Treatment

Mean GUSB activity in control brains (nmol/h/mg)

GUSB activity per slide (eight transverse sections, 320-µm rostral–caudal dimension) (nmol/h/mg) Slide A

Slide B

Slide C

Slide D

Slide F

1

Normal

None

2

Normal

None

7.3

3

Normal

None

10.1

3931

MPS VII

None

0.0

3974

MPS VII

None

0.1

6154

MPS VII

GUSB-JS

0.3

0.4

0.6

1.0

0.6

6170

MPS VII

GUSB-JS

0.7

0.8

1.1

1.2

1.0

6197

MPS VII

GUSB-JS

0.6

1.3

2.5

3.7

1.7

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9.2

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METHOD ated at the LAT RNA start site, proceeding through the GUSB cDNA and 2-kb LAT intron and ending at the mLAT poly(A) signal (7.6 kb from the start site in the intact locus). The 0.9-kb difference in length of the large transcript from the different viruses reflects the 0.9-kb BstEII–BstEII deletion present in the GUSB-CD virus (Fig. 3). The prominent expression of polyadenylated mLAT transcripts is unusual. In wild-type HSV-1, the stable 2-kb LAT intron can be readily detected in latently infected ganglia by Northern blot analysis (8, 59–61), but the 8.3kb primary transcript mLAT is barely detectable by Northern blot, both in vivo and in vitro (11, 49). Evidence for a low abundance large transcript comes from more sensitive approaches such as in situ hybridization and RNase protection assays, using probes covering both 5′ and 3′ sequences outside the 2-kb LAT (9, 11, 43, 62, 63). Our data suggest that insertion of the 2.4-kb GUSB cDNA at the 5′ end of the LAT locus stabilized the LAT-GUSB primary transcript. Increased stability of mLAT primary transcripts has also been seen in latently infected cells with a vector having the lacZ gene fused to the 5′ end of the LAT primary transcript and inserted at an ectopic site in the viral genome (27). The detection of abundant GUSB enzyme activity in MPS VII mouse brains latently infected with virus GUSB-JS also strongly supports the hypothesis that a stable polyadenylated LAT exon RNA is being expressed. We previously found that genetically diseased MPS VII mice were more susceptible to viral infection than normal animals (47). However, nonpathogenic HSV-1 strains such as 1716 (34) can be safely used for intracranial inoculation in these animals (47, 60). This virus has a deletion of 759 bp in the ICP-34.5 gene, the product of which is involved in inhibition of translation shutdown during acute infection. Viruses with this deletion have been shown to be unable to overcome the host cellinduced translational block mediated by PKR phosphorylation of translation initiation factor elF2α and thus shut off viral protein synthesis prematurely (64, 65). This effect is more pronounced for late viral gene products and, since the LAT promoter is expressed with late kinetics in replicating cells during acute infection, it might be expected to reduce the GUSB activity detected during acute infection. In addition, ICP-34.5 maps to the mLAT region of the genome and is coded on the opposite strand from LAT, but it is not expressed during latent infection. Thus, deletion of this sequence might affect the ability of a HSV-1 vector to form latent infections or express the LAT gene during latency. However, the detection of abundant mLAT transcripts on Northern blots (Fig. 3) and measurable amounts of GUSB enzyme activity in latently infected mouse brain (Table 2) indicate that this was not the case. In our vectors, the LAT promoter has been left in its natural site in the viral genome with no sequence modification of the upstream promoter elements. The vector

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with a substitution of the GUSB cDNA for HSV sequences at the LAT exon 1 2-kb intron boundary (GUSB-CD), which removed the splice donor region, was less effective than either of the vectors in which the intron was left intact. Previously we found that this design resulted in a significantly decreased number of cells in which latency was established (10). These data suggest that either the LAT intron or the exon sequences surrounding it are important for vector functioning. Insertion of the cDNA at the 5′ end of exon 1 resulted in a vector (GUSB-JS) that expressed GUSB in a much greater number of cells than with the insertion at the 3′ end of the exon (GUSB-JF). In contrast, the patterns of in situ hybridization were similar, which suggests that a translational difference may account for the difference in expression with the position in exon 1. This was similar to another vector which expressed β-gal from the LAT promoter, where we found that β-gal activity did not correspond to the β-gal RNA levels in mouse trigeminal ganglia following peripheral inoculation and in which sequences in LAT exon 1 were implicated (55). The genomic structures of recombinant viruses GUSB-JF and GUSB-JS are the same except that the 371-bp StyI–StyI sequence is present upstream of the translation initiation site of GUSB cDNA in virus GUSB-JF but is downstream of the entire GUSB cDNA in the virus GUSB-JS. This 371-bp LAT sequence contains an ATG that is out of frame with the GUSB ATG in the JF vector. Thus, different levels between transcription and GUSB activity in the JF virus could be accounted for by translation being initiated from the upstream LAT ATG preventing use of the GUSB ATG. Although a protein encoded by LAT has not been identified, our data suggest that a LAT exon 1 ATG may be functional. Although vector viruses were injected into the caudate putamen, most of the cells positive for GUSB activity by the histochemical reaction were found in the thalamus and hypothalamus. This and the fact that positive cells were present in both the ipsilateral and contralateral sides of the unilaterally injected brain indicate that significant amounts of axonal transport occurred. Previously we showed that infection with the parental virus (1716) into the caudate/putamen resulted in expression of LAT in the diencephalon (51). However, significant amounts of LAT-positive cells were also seen in the neocortex, which was not observed in the present experiments. This may indicate that insertion of a cDNA into the LAT locus changed the properties of the virus. The major connections in this region involve projections from the caudate to the thalamic areas, which suggests that some anterograde movement may have occurred. Anterograde movement of HSVs also appears to occur from peripheral sites of inoculation because the vectors move retrograde to ganglion cells (trigeminal) and then into the brain stem, which is believed to follow anterograde projections from the ganglion cell bodies (32). However, the volumes of virus injected in our

MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy

METHOD experiments (1 µl) were too large to map the movement of the vectors, which requires nanoliter amounts and precision placement (66). The levels of GUSB expressed after latency was established were significantly higher than in previous studies using HSV vectors (32, 33). Using other gene transfer and transplantation methods, we have estimated that about 1–5% of normal GUSB levels in the brain will be required to effect a change in the storage phenotype (46, 67). Although this was achieved in some regions, it will be necessary to achieve wide distribution of the normal enzyme to treat the global brain lesions that occur in this and other lysosomal storage diseases. The ability of HSV to move transneuronally suggests, however, that it may be possible to target specific areas of the brain for correction. Use of this approach will depend on improved understanding of the nature of the movements of these vector viruses within cells and between structures in the brain. The development of a vector virus that expresses high levels of GUSB during latency will now allow the study of various pathways, as well as the therapeutic potential in MPS VII animals. Although there was insufficient material to perform pathology studies in this set of experiments, the levels of GUSB expression seen with the GUSB-JS vector were comparable to those of other vectors that have been shown to correct storage lesions, indicating that it should be effective in future therapeutic trials. This vector also should be a useful design for expressing other genes of interest to manipulate neurons in targeted regions of the CNS. ACKNOWLEDGMENTS We are indebted to Tara Friebel, Vikram Suri, Micheal Parente, Aracelis Polesky, and Colleen Jones for excellent technical assistance. This work was supported by National Institutes of Health Grant NS 29390.

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MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright  The American Society of Gene Therapy