NO20025657L - Use of Baculovirus Vectors in Gene Therapy - Google Patents

Description

Field of the invention

The present invention relates to gene delivery using a viral vector.

Background for the invention

Efficient gene transfer would be a beneficial tool for the treatment of vascular diseases such as post-angioplasty restenosis, post-bypass atherosclerosis, peripheral atherosclerotic disease, stenosis of vascular prosthetic anastomoses and thrombus formation. Various techniques have been developed for this purpose; see, for example, Yla-Herttuala et al, J. Clin. Invest. 95:2692-8 (1995) and Laitinen et al., Hum. Gene. Ther. 8:1645-50 (1997).

WO-A-98/20027 describes a periadventitial collar that can be used for arterial gene transfer during vascular surgery. However, there is a continuing need for simpler and more efficient gene transfer vectors. Only a temporary expression of the transgene may be necessary to achieve a beneficial biological effect in cardiovascular applications; see Yla-Herttuala et al, Lancet 355:213-222 (2000).

Baculoviruses have long been used as biopesticides and as tools for efficient recombinant protein production in insect cells. They are generally considered safe, because of their natural high species specificity and because they are not known to propagate in any non-invertebrate host. Although the virions have been shown to enter certain cell lines derived from vertebrate species, there is no evidence that viral gene expression has been detected using natural viruses. However, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), containing an appropriate eukaryotic promoter, can transfer and express target genes efficiently in many mammalian cell types; see, for example, Hofmann et al, PNAS USA 92:10099-10103 (1995). In addition, Barsoum et al., Hum. Gen. Ther. 8:2011-8 (1997) stated that baculovirus having the vesicular stomatitis virus C glycoprotein in its envelope significantly increases the efficiency of transduction of human hepatoma cell lines and expands the range of mammalian cell types that can be transduced by baculovirus. Stable transduction of mammalian cells by baculovirus has been achieved by either including an expression cassette encoding a dominant selectable marker in the baculovirus genome or by using a hybrid baculovirus-adeno-associated virus vector; see Condreay et al, PNAS USA 96:127-132 (1999) and Palombo et al, J. Virol. 72:5025-34 (1998).

Sandig et al, Hum. Gene Ther. 7:1937-45 (1996) reported unsuccessful attempts to use baculovirus for in vivo gene delivery to mice and rats by systemic or intraportal application as well as by direct injection into liver parenchyma. One reason for this is presumably inactivation of the baculovirus by the classical pathway of the serum complement system.

WO-A-00/05394 describes baculovirus vectors and their use for gene transfer into vertebrate nerve cells.

Summary of the invention

It has now been found that inactivation of baculovirus can be avoided. In particular, it has been shown that baculovirus can mediate periadventitial gene transfer into rabbit carotid arteries with an efficiency comparable to adenovirus. Ease of manipulation and rapid construction of recombinant baculoviruses, their lack of cytotoxicity in mammalian cells even at a high multiplicity of infection, their natural inability to replicate in mammalian cells and their large capacity for insertion of foreign sequences make baculoviruses a very suitable tool for in in vivo gene therapy.

The present invention can exploit the advantageous properties of baculovirus, in a suitable vector from which the gene is expressed, if administered (in or ex vivo) to a place on the body where there is no blood or which is essentially free of blood. Thus, periadventitial or, more specifically, collar-mediated local gene delivery allows gene transfer essentially in the absence of serum, thus avoiding deleterious effects of serum components. The new method also avoids two other main problems encountered in systemic gene delivery, i.e. a rapid redistribution of the virus from the injection site and a drop in the local concentration of the virus.

Description of the present invention

Suitable delivery systems, active materials, preparations, doses, etc., are illustrated in WO-A-98/20027 and also WO-A-99/55315 (the contents of which are incorporated herein by reference). Thus, by way of example only, the delivery constituent may be a collar or a cover. Compared to the publications, the gene delivery vector is a baculovirus.

Baculoviruses are of course known and those skilled in the art will be able to construct any suitable vector for use in the present invention. It will also be apparent that the broad knowledge of baculovirus biology and AcMNPV genomes will aid the construction of improved second-generation viruses for gene transfer applications. Simplicity of construction and capacity to accept large foreign DNA fragments (>20 kbp), allows the development of baculoviruses that have enhanced or targeted cell tropism along with more stable, temporal and cell type-specific control of transgene expression. A recombinant baculovirus for use in the invention can be formulated in a drug for therapeutic use in a known manner.

Routes and sites of administration of the invention include intraocular application, intra-articular application, surface intradermal application, ureters, bladder, fallopian tubes, gall bladder, spinal cord,

cerebrospinal fluid chamber, the pleural cavity and the intraperitoneal cavity. Sites that have been used (see the examples) are arteries, brain and skeletal muscle, including, for example, mycocytes, satellite cells and regenerating myoblasts. Gene delivery can be performed by direct injection or by different types of catheters.

If appropriate, body parts can be rendered "bloodless" during surgery. This technique is often used in leg or arm surgery by tightening tight pressure around the arm or thigh, thus preventing blood flow. The body part can then be perfused with saline to remove blood, and baculovirus transfection can then be performed.

The invention can be used for the delivery of an agonist for a VEGF receptor, e.g. described in more detail in WO-A-98/20027. Furthermore, with a suitable choice of gene, it can be used in the treatment of cancer, e.g. in the brain.

A further aspect of the present invention relates to graft organs and vessels which can be perfused with saline ex vivo and subjected to ex vivo baculovirus injection.

The following experimental work illustrates the invention.

Example 1

Production of recombinant baculoviruses

Viruses were constructed using the transfer vector pFASTBad (pFB) (Gibco BRL, Life Technologies, Gaithersburg, MD, USA). A nuclear targeting /3-galactosidase ((3nt-Gal) cassette with a cytomegalovirus (CMV) promoter was inserted into the Sful site of pFB in reverse orientation with respect to the polyhedrin promoter, to form the plasmid pFBCMV-j3nt-

Recombinant virus was generated using a BacTo-Bac™ baculovirus expression system (Gibco BRL). Viruses were amplified in Spodoptera frugiperda 9 (Sf9) suspension cultures (SF-900 medium, Gibco BRL) for 3 days using a cell density of 2 x 10<6> cells/ml. For 50 ml culture, 200 µl of primary transfection supernatant was used as inoculum. To obtain 1 liter of amplified virus, 2 ml of amplified virus stock was used as inoculum. The cell culture medium was centrifuged at 16,000 g for 20 min at room temperature to remove cell debris. The clarified supernatant was transferred to ultracentrifuge tubes lined with 1.5 ml of 25% sucrose in phosphate-buffered saline (PBS) and virus was concentrated by centrifugation (120,000 g. 4EC, 1.51). The virus pellet was resuspended in 35 ml of ice-cold PBS , transferred to ultracentrifuge tubes containing 3 ml of 25% sucrose in PBS and centrifuged as above. Final virus pellet was resuspended in 10 ml of cold PBS, filtered through 0.45 ixm filter and kept at 4EC protected from light for further use. Virus titer was determined by a plaque assay on Sf9 cells The virus preparations were analyzed for lipopolysaccharide and bacteriological contaminants.

Production of recombinant adenoviruses

Nuclear-targeted LacZ encoding adenovirus (pCMVnls/acZAd5) was constructed and prepared as described by Laitinen et al, supra. Virus preparations were analyzed for replication competent virus, lipopolysaccharide and bacteriological contaminants as described by Laitinen et al, Hum. Gen Ther. 9:1481-6(1998).

Gene transfer in vitro

Rabbit aortic cells (RAASMC; Yla-Herttuala et al (1995), supra) and human carcinoma/endothelial cell-like ECV-304 cells (ATTC CRL-1998) were plated at a density of 10,000 cells per well. well (Falcon Culture Slide, Becton Dickinson, Meylan, France). Cells were allowed to attach for 3 hours before transduction in serum-free medium (DMEM, 100 units/ml penicillin and 100 µg/ml streptomycin, Gibco BRL). Viruses were added to medium at MOI of 200 or 1000 and cells were incubated for 90 min. at 37EC. After transduction, growth medium containing 10% fetal bovine serum was added either with or without 10 mM n-butyric acid (Sigma, St. Louis, MD, USA). After 18 hours of incubation, the media were removed and the cells were washed three times with PBS. The cells were fixed with 1.25% glutaraldehyde for 15 min. and washed three times with PBS. X-Gal (MBI Fermentas, Lithuania) labeling solution (1 mg/ml, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 x PBS) was added to the cells and incubated for 3 h at 37EC. The cells were then washed with PBS and further fixed with 4% paraformaldehyde (PFA) for 10 min. After washing with PBS, the cells were counterstained with Mayer's Carmalum for 5 min. Blue X-Gal-positive cells were counted and the transduction efficiency was expressed as the percentage of positive cells out of the total number of cells.

ONPG trial

/acZ-encoded /?-galactosidase activity in transduced RAASMC cells was measured using a colorimetric substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma), as described by Ruponen et al, Biochim. Biophys. Acta 1415:331-41 (1999).

In vitro toxicity tests

Cells were plated in 96-well plates at a density of 20,000 cells per well. well in 100 µl growth medium consisting of DMEM with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). Virus transductions were performed as for the transduction efficiency experiment and cells were incubated for 48 hours at 37EC. Growth medium was removed and cells were washed with PBS. Serum-free DMEM without phenol red, containing MTT solution (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide, 5 mg/ml, final concentration 0.3 mg/ml) was then added and the cells were incubated for 2 h. To dissolve the dark blue formazan crystals, MTT solution was removed, 150 µl of 1 M DMSO was added to each well and mixed thoroughly. Absorbance diaper needle at 570 nm. Survival percentage was calculated compared to absorbance of non-virus or non-butyrate wells (100% survival).

Animal testing

New Zealand white (NZW) male rabbits [ n = 12; 2.8-3.7 kg) was used. Fentanyl-fluanisone (0.3 ml/kg, s.c.; Janssen Pharmaceutica, Beerse, Belgium) and midazolam (1.5 mg/kg, i.m., Roche, Basel, Switzerland) were used for anesthesia. The left and right carotid arteries were exposed using a midline neck incision. The artery was carefully separated from the surrounding tissue and a 3 cm long soft plastic "silastic" collar (see Laitinen et al (1997), supra) was placed around it. The rabbits were anesthetized again for gene transfer, which was performed 5 days after installation of the collar in exactly the same manner as described by Laitinen et al (1997), supra, who compared transfection efficiency of plasmid/liposomes, pseudotyped retrovirus and adenovirus. The wells were opened and filled with 500 µl of the gene transfer solution containing 1 x 10<9>pfu of adenovirus or baculovirus. For each animal, the left carotid artery was used for adenovirus and the right carotid artery for baculovirus treatment. Four rabbits were sacrificed 3, 7 and 14 days after the gene transfer and the arteries were removed for histological analyses. All animal procedures were approved.

Histological analysis

Collared arteries were divided into three equal parts: the proximal third was immersion-fixed in 4% PFA/15% sucrose (pH 7.4) for 4 h, rinsed in 15% sucrose (pH 7.4) overnight and embedded in paraffin. The medial part was immersion-fixed in 4% PFA/PBS (pH 7.4) for 30 min, rinsed in PBS (pH 7.2) and embedded in OCT compound (Miles Scientific, Naperville, IL, USA). The distal part was snap-frozen in liquid nitrogen and stored at -70EC for mRNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR).

Randomly selected frozen sections (10/m) from each rabbit were labeled with X-Gal (MBI Fermentas) for 18 hours to identify β-galactosidase positive cells. Gene transfer efficiency was calculated as X-Gal-positive cells per mm<2> of adventitia or whole artery wall in eight randomly selected sections by two independent observers. Mean values V standard deviation of the mean (SEM) for the results are indicated. Paraffin sections were used for immunocytochemical detection of endothelium (CD-31; 1:50 dilution; Dako, Hamburg, Germany), macrophages (RAM-11; 1:100 dilution; Dako), smooth muscle cells (HHF-35; 1:50 dilution; Dako) and T cells (MCA-805; 1:100 dilution; Dako) as described by Yla-Herttuala (1995), supra. Controls for the immunolabeling included sections where the first antibody was omitted and sections incubated with class- and species-matched immunoglobulins. Morphometry and image analysis were performed using hematoxylin-eosin-stained paraffin sections and Image-Pro Plus™ software with an Olympus AX70 microscope (Olympus Optical, Japan).

RT-PCR

Total RNA was extracted from transduced carotid artery segments using Trizol reagent (Gibco-BRL) and treated with excess RQ1 RNAse-free DNAse (Promega, Madison, WI, USA). M-MulV reverse transcriptase (MBI Fermentas) was used for cDNA synthesis. Primers (20 pmol each) for /acZ were designed to distinguish between endogenous and transduced genes by selection of the 5' primers from the CMV promoter and the 3' primers from the coding region. Dynazyme polymerase (Finnzymes, Espoo, Finland) was used for amplification.

For /acZ amplification, the primers were SEQ ID NO:1 for adenovirus (A) and SEQ ID NO:2 for baculovirus (B) as forward primers and SEQ ID NO:3 for both as a reverse primer. Warm start (95EC 5 min; 58EC 3 min) was followed by 39 cycles, each consisting of 95EC 1 min, 58EC 2 min, 72EC 3 min with the final extension 10 min at 72EC. 5 µl of the first PCR product was used for the second PCR with forward primers SEQ ID NO:4 (A) and SEQ ID NO:5 (B). The reverse primer for both was SEQ ID NO:6. The first PCR cycle was 95EC for 5 min. followed by 20 cycles as in the first PCR.

Gene transfer in vitro

To test baculovirus stocks, RAASMC and ECV-304 cells were transduced at a multiplicity of infection (MOI) of 200 or 1000 pfu per cell in the absence or presence of 10 mM sodium butyrate and the percentage of X-Gal positive cells was counted. The results were compared with cells transduced with /acZ adenovirus under identical conditions (Table 1). Consistent with published results, (Condreay et al, supra) addition of butyrate to cell cultures remarkably increased the expression of the transgene, particularly with baculovirus. The RAASMC cells appeared to be more susceptible to baculovirus transduction (91% infected at MOI 1000) than ECV-304 cells (21% infected at MOI 1000). Levels of β-galactosidase activity in the RAASMC cells were also measured by a quantitative biochemical assay with o-nitrophenyl-β-D-galactopyranoside (ONPG). The results were consistent with X-Gal labeling showing an increase in transgene expression after butyrate treatment in the baculovirus-transduced cells.

In vitro toxicity

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the cytotoxicity of the virus preparations (Table 2). Neither baculovirus nor adenovirus showed any significant cytotoxicity to RAASMC cells in the absence of butyrate at MOI 200 or 1000. However, together with butyrate, some cytotoxicity was detected in these cells with baculovirus at an MOI of 1000. Similar results were obtained with primary WHHL- ( Watanabe hereditary hyperlipidemic) rabbit fibroblasts except that no cytotoxic effects were detected for baculovirus in the presence of butyrate at an MOI of 1000 (data not shown).

Gene transfer in vivo

To determine whether baculovirus can be used for in vivo gene transfer, NZW rabbits were euthanized 3, 7 and 14 days after gene transfer. Due to nuclear targeting of β-galactosidase expression, intense X-Gal labeling was localized in the nuclei of the transduced cells. The number of cells positive for β-galactosidase activity was calculated from baculovirus-transduced

(1 x 10<9>pfu per artery) carotid arteries and were compared with similarly treated adenovirus-transduced arteries. Both viruses mediated delivery of the marker gene to the vessel wall. The number of transgene-positive cells for baculovirus- and adenovirus-treated arteries on day 3 was 12 V 5 and 23 V 7/3-galactosidase-positive cells/mm<2>adventitia, respectively (11 V 4 and 19 V 6/mm<2> of the entire arterial wall). On day 7, the corresponding values were 17 V 6 and

22 V 7 (15 V 5 and 18 V 6). On day 14 the values were 0.1 V 0 and 0.2 V 0.1 (0.1 V 0 and 0.1 V 0.1). Thus, baculovirus-mediated gene expression was transient with a similar efficiency and time pattern to that of adenovirus-mediated gene transfer. Transgene expression in the arterial wall was also verified with

RT-PCR.

The mean value of intima/media ratio (all arteries) was 0.18 V 0.03 for baculovirus- and 0.12 V 0.01 for adenovirus-treated arteries, indicating that the methods do not damage the vessel wall. Histology of arteries 7 days after gene transfer detected no β-galactosidase activity outside the adventitia. For both viruses, macrophage infiltrates and some T cells were detected in the transduced arteries by RAM-11 and MCA-805 immunolabeling, respectively. The arterial structure and endothelium remained intact throughout the experiments. Histological findings at all time points are summarized in Table 3.

Example 2

By using essentially the same procedure as in Example 1, it has been shown that baculovirus gene transfer also works on brain and skeletal muscle. When using baculovirus//acZ, the rat home shows positive transfection in different types of brain cells, especially in choroid plexus cells in ventricles and endothelial cells. The profile of transfected cells is clearly different from that of adenovirus.

Furthermore, baculovirus transfection has been demonstrated in rabbit skeletal muscle. Baculovirus encoding lacZ (1.8 x 10<10>PFU) was directly injected into the adductor muscle of NZW rabbits via a 25 G needle. The injection volume was 0.5 ml. Tissue samples were collected 7 days after the gene transfer and X-Gal labeling was performed overnight. These results clearly indicate that baculovirus can be used for transfection of many cell types in mammals, i.e. not only arterial cells.

The accompanying drawing illustrates the construction of a nuclear-targeting/3-galactosidase-encoding baculovirus transfection cassette. IN

in principle, this is a standard common baculovirus with polyhedrin promoter, into which restriction sites and CMV-NT/acZ expression cassette have been cloned. /acZ expression cassette is oriented opposite the polyhedrin promoter. The sequence of the CMV-nt /acZ expression cassette is in SEQ ID NO:7.

Claims (11)

1. Method for delivering a gene product, wherein the gene is provided in a baculovirus vector and the vector is introduced into a body space free or usually free of blood. 2. Method according to claim 1, wherein the delivery is periadventitial. 3. Method according to claim 1 or claim 2, where the gene product is an agonist for a VEGF receptor. 4. A method according to any one of the preceding claims, for the treatment of intimal hyperplasia. 5. Method according to claim 1 or claim 2, wherein the compartment comprises arterial cells. 6. Method according to claim 1 or claim 2, where the space comprises brain cells. 7. Method according to claim 1 or claim 2, where the space comprises skeletal muscle. 8. Device for periadventitial delivery of a gene product, where the gene is delivered in a baculovirus vector from which the gene can be expressed. 9. Device according to claim 8, in the form of a collar. 10. Device according to claim 8, in the form of a cover. 11. Device according to any one of claims 8 to 10, wherein the gene product is an agonist for a VEGF receptor.