KR20030072213A - Use of baculovirus vectors in gene therapy - Google Patents

Abstract

In a method for delivering a gene product, the gene is provided to a baculovirus vector, which vector is applied to a body compartment with little or no blood. In a device for perivascular delivery of a gene product, the gene is provided to a baculovirus vector in which the gene can be expressed.

Description

Use of baculovirus vectors in gene therapy {USE OF BACULOVIRUS VECTORS IN GENE THERAPY}

Effective gene delivery is a useful tool in the treatment of vascular diseases such as post-angiogenic restenosis, post-tubular atherosclerosis, peripheral atherosclerosis, stenosis of angioplasty, and thrombus formation. Various techniques have been developed for this purpose; See 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 discloses a perivascular collar that can be used for arterial gene transfer during vascular surgery. However, there is a continuing need for easier and more efficient gene transfer vectors. Only transient expression of the transgene may be required to achieve useful biological effects in cardiovascular applications; See Yla-Herttuala et al , Lancet 355: 213-222 (2000).

Baculoviruses have long been used as a means for producing recombinant proteins and as biopesticides useful in insect cells. They are generally considered safe because of their inherently high species selectivity and because they are known to not proliferate in non-invertebrate hosts. Although virions appear to be inserted into certain cell lines derived from vertebrate species, there is no evidence of viral gene expression detected using native viruses. However, Autographa californica multinuclear polyhediovirus virus (AcMNPV), which contains a promoter of appropriate eukaryotic cells, can effectively transplant and express target genes in various mammalian cell types. have; For reference, see Hofmann et al, PNAS USA 92: 10099-10103 (1995). Moreover, Barsoum et al, Hum. Gene. Ther. 8: 2011-8 (1997), a baculovirus having an vascular stomatitis virus C glycoprotein in the envelope significantly increases the efficiency of transduction of human liver cancer cell lines and can be transduced by baculovirus. It has been reported to broaden the cell type range of mammals. Stable transduction of mammalian cells by baculovirus involves expression of a cassette encoding a potent selective marker into the baculovirus genome or a hybrid baculovirus-adeno-associated virus Complete with the use of vectors; 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) report a failed attempt to use baculovirus for in vivo gene delivery to mice and rats, not only by direct infusion into hepatic parenchyma but also by systemic or portal application. It was. One reason for the failure is probably the inactivation of baculovirus by the typical route of serum supplementation system.

WO-A-00 / 05394 discloses baculovirus vectors and their use for delivering genes to neurons of vertebrates.

The present invention relates to gene transfer using viral vectors.

1 shows the construction of a nuclear-targeting β-galactosidase-encoding baculovirus transfection cassette.

Summary of the Invention

It is now known that inactivation of baculovirus can be avoided. In particular, baculoviruses are known to modulate perivascular gene delivery to rabbit carotid arteries with efficiencies comparable to adenoviruses. Ease of manipulation and rapid construction of recombinant baculoviruses, their cytotoxic deficiency in mammalian cells with high complexity of infection, their inability to replicate in their mammalian cells, and wide capacities for insertion of their heterologous sequences Makes baculovirus a very suitable tool for in vivo gene therapy.

The present invention can take advantage of the beneficial properties of baculovirus in a suitable vector, where the gene is expressed, if administered (in vivo or ex vivo) to a body part without blood or essentially blood free. Therefore, around the outer membrane or more specifically, color-regulated local gene delivery necessarily enables gene transfer in the absence of serum, thus avoiding the deleterious effects of serum components. The novel method also avoids two other major problems encountered in systemic gene transfer such as, for example, rapid redistribution of the virus from the site of injection and a drop in local concentration of the virus.

Description of the invention

Suitable delivery systems, active materials, formulations, single doses and the like are shown in WO-A-98 / 20027 and WO-A-99 / 55315. Thus, by way of example only, the delivery vehicle may be a collar or a wrap. Compared with this publication, the vector for gene delivery is baculovirus.

Baculoviruses are well known, of course, and those skilled in the art will be able to construct vectors suitable for use in the present invention. It will also be apparent that well known information of the baculovirus biology and AcMNPV genome will assist in the regulation of advanced second generation viruses for gene transfer applications. The ease of construction, and the large heterologous DNA-segment (> 20 kbp) capacity, together with the more stable, transient, cell type-specific regulation of transgene expression, expand or target the cell fragility of the baculovirus To grow. Recombinant baculovirus for use in the present invention may be formulated in a known manner, as a medicament for therapeutic use.

Routes of administration and sites of the invention include intraocular application, intraarticular application, appearance in the skin, ureters, bladder, palopioducts, gallbladder, spinal cord, cerebrospinal fluid compartments, pleural cavities and cavities in the abdominal cavity. Sites used are arteries, brain and skeletal muscles, including, for example, mucous cells, concomitant cells and regenerative source cells. Gene delivery can be through direct injection or through various types of catheters.

If appropriate, the body part can be made "blood free" during surgery. This technique is often used in foot or arm surgery by injecting tight pressure around the arm or thigh to prevent blood flow. The body part can then be perfused with saline to remove blood, and then a baculovirus infection can occur.

The present invention can be used for the delivery of agonists of VEGF receptors, for example as disclosed in more detail in WO-A-98 / 20027. In addition, appropriate selection of genes can be used for the treatment of cancer, for example in the brain.

Another aspect of the invention relates to transplanting organs and blood vessels, which can be perfused with saline in Ex vivo and are infected with baculovirus in Ex vivo.

The invention is illustrated by the following experiment.

Example 1

Preparation of recombinant baculovirus

Virus was constructed using the transfer vector pFASTBac1 (pFB) (GibcoBRL, Life Technologies, Gaithersburg, MD, USA). Nuclear targeting β-galactosidase (β _nt -Gal) cassettes with a cytomegalovirus (CMV) promoter were inserted into Stu l of pFB against the polyhedrin promoter, resulting in plasmid pFBCMV-β _nt .

Recombinant virus was produced using the BacTo-Bac ™ baculovirus expression system (Gibco BRL). The virus was amplified at 2x10 ⁶ cells / ㎖ using the cell concentration of the spokes FER help TB program rugi for 3 days (Spodoptera frugiperda) 9 (Sf9) suspension culture medium (SF-900 medium, Gibco BRL). For 50 ml of culture, 200 μl of initial transfection supernatant was used as the inoculum. The cell culture medium was centrifuged at 16,000 g for 20 minutes at room temperature to remove cell debris. The purified supernatant was transferred to an ultracentrifuge tube with 1.5 ml of phosphate-buffered saline (PBS) containing 25% sucrose and the virus condensed by centrifugation (120,000 g, 4 ° C., 1.5 hours). Virus pellets were resuspended in 35 ml of ice-cold PBS, transferred to an ultracentrifuge tube containing 3 ml of PBS containing 25% sucrose and centrifuged as above. The final virus pellet was resuspended in 10 ml of cooled PBS, filtered with a 0.45 μm filter and stored at 4 ° C. with light blocking for later use. Virus titers were determined by plaque assay in Sf9 cells. Virus preparations were analyzed for contaminants of lipopolysaccharide and bacteriology.

Preparation of Recombinant Adenovirus

Nuclear target LacZ encoding adenovirus (pCMVnls lac ZAd5) is described in Laitinen et al. It was constructed and prepared as disclosed by. Virus preparations are described in Laitinen et al , Hum. Gene Ther. 9: 1481-6 (1998) as analyzed for replication-capable viruses, lipopolysaccharides and bacteriological contaminants.

In vitro gene transfer

Rabbit aortic cells (RAASMC above; Yla-Herttuala et al , (1995)) and human carcinoma / endothelial cell-like ECV-304 cells (ATTC CRL-1998) were used in the wells (Falcon Culture Slide, Becton Dickinson, Meylan, France). Plated at a density of 10,000 cells per cell. Cells were attached for 3 hours in serum-free medium before transduction (DMEM, 100 units / ml penicillin and 100 μg / ml streptomycin, Gibco BRL). Virus was added to the medium at 200 or 1000 MOI and cells were incubated at 37 ° C. for 90 minutes. After transduction, growth medium containing 10% fetal bovine serum was added with or without 10 mM of n-butyric acid (Sigma, St. Louis, MD, USA). After 18 hours of incubation, the medium was removed and the cells washed three times with PBS. Cells were fixed for 15 minutes with 1.25% glutaraldehyde and washed three times with PBS. X-Gal (MBI Fermentas, Lithuania) coloring solution (1 mg / ml, 2 mM MgCl ₂ , 5 mM K ₃ Fe (CN) ₆ , 5 mM K ₄ Fe (CN) ₆ , 1 × PBS) was added to the cells and 3 at 37 ° C. Incubate for hours. Cells were then washed with PBS and further settled with 4% paraformaldehyde (PFA) for 10 minutes. After washing with PBS, cells were counted-stained for 5 minutes with Mayer Carmalum. Blue X-Gal positive cells were counted and transduction efficiency was expressed as percentage of positive cells to total cell number.

ONPG Evaluation

LacZ-encoded β-galactosidase activity in transduced RAASMC cells is described by Roponen et al , Biochim. Biophys. Acta 1415: 331-41, as described in (1999), the colorimetric substrate, o - was measured using a nitrophenyl β-D- galacto-pyrano seed (o -nitrophenyl β-D-galactopyranoside , ONPG, Sigma).

In vitro Toxicity Assessment

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

Animal experiment

New Zealand White (NZW) rabbit males (n = 12; 2.8-3.7 kg) were used. Fentanyl-fluanison (0.3 mL / kg, sc; Janssen Pharmaceutica, Beerse, Belgium) and midazolam (1.5 mg / kg, im, Roche, Basel, Switzerland) were used for anesthesia. The right and left carotid arteries were exposed using midline neck incisions. The artery is carefully separated from the surrounding tissue, and a 3 cm long elastic collar (see Laitinen et al (1997) above) is located around it. Rabbits were subjected to gene transfer, performed exactly 5 days after the device of the collar, in the same manner as described by Laitinen et al (1997), comparing the transfection efficiency of plasmids / liposomes, gastric retroviruses and adenoviruses. Anesthetized for The collar was opened and filled with 500 μl of gene transfer solution containing adenovirus or baculovirus 1 × ^{10 9} pfu. In each animal, the left carotid artery was used for adenovirus and the right carotid artery was used for baculovirus treatment. Four rabbits were killed 3, 7, 14 days after gene transfer and the carotid artery was removed for histological analysis. All animal processes have been approved.

Histological Analysis

The detached artery was divided into three parts of equal size. The third near the base was soaked in 4% PFA / 15% sucrose (pH 7.4) for 4 hours, washed overnight in 15% sucrose (pH 7.4) and planted in paraffin. The middle portion was soaked in 4% PFA / PBS pH 7.4 for 30 minutes, fixed with PBS pH 7.2 and planted in OCT compounds (Miles Scientific, Naperville, IL, USA). The terminal portion was quenched in liquid nitrogen and stored at −70 ° C. for mRNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR).

Randomly selected frozen sections (10 μm) from each rabbit were stained for 18 hours with X-Gal (MBI Fermentas) to identify β-galactosidase positive cells. Gene transfer efficiencies were calculated as X-Gal positive cells per mm 2 of the outer membrane or by the walls of the total arteries in eight randomly selected sections by two independent observers. The mean value ± standard error of the mean (SEM) of the above result was created. Paraffin sections include endothelial (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, above, for immunocytochemical detection. The comparative group for immunostaining included fragments in which the first antibody was omitted, and the sections were incubated with cavities and species comparable to immunoglobulins. Anthropometric and phase analysis was performed using hematoxylin-eosin-pigmented paraffin sections and Olympus AX70 microscope (Olympus Optical, Japan) and Image-Pro Plus ™.

PT-PCR

Total RNA was extracted from transduced carotid artery segments using Trizol Reagent (Gibco-BRL) and treated with DNAse (Promega, Madison, WI, USA) without excess RQ1 RNAse. M-MulV reverse transcriptase (MBI Fermentas) was used for cDNA synthesis. Primers for lacZ (20 mmol each) were designed to distinguish between resistant and transformed genes by selecting 5 'primers from the CMV promoter and 3' primers from the coding region. Dynazyme polymerase (Finnzymes, Espoo, Finland) was used to amplify.

For lacZ amplification, the primers used SEQ ID NO: 2 for adenovirus (A) and SEQ ID NO: 2 for baculovirus (B) as forward primers, and SEQ ID NO: 3 for reverse primers. Starting at high temperature (95 ° C., 5 minutes; 58 ° C. 3 minutes), 39 cycles consisting of 95 ° C. 1 minute, 58 ° C. 2 minutes, and 72 ° C. 3 minutes were repeated and finally continued at 72 ° C. for 10 minutes. 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). Both reverse primers were SEQ ID NO: 6. The first PCR cycle was repeated 5 minutes at 95 ° C. followed by 20 cycles as in the first PCR.

In vitro gene transfer

To test baculovirus stocks, RAASMC and ECV-304 cells were transduced at 1000 or 200 pfu of infection (MOI) per cell with or without 10 mM sodium butyrate, and X-Gal Positive cells were calculated in percent. The results were compared with cells transduced with lacZ -adenovirus under the same conditions (Table 1). In line with the published results, the addition of butyrate to the cell culture (Condreay et al , supra) significantly increased the expression of baculovirus and transgene, in particular. RAASMC cells appeared to be more susceptible to baculovirus transduction (91% infected at MOI 1000) than ECV-304 cells (21% infected at MOI 1000). The grade of β-galactosidase activity in RAASMC cells was also determined by quantitative biochemical evaluation using o-nitrophenyl β-D-galactopyranoside (ONPG). This result was consistent with X-Gal staining showing an increase in transgene expression after butyrate treatment in baculovirus-transduced cells.

In vitro toxicity

3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltedrazolium bromide (MTT) -assessment was used to measure the cytotoxicity of the viral preparation (Table 2). None of the baculovirus or adenovirus showed any major cytotoxicity to RAASMC cells in the absence of butyrate at MOI 200 or 1000. However, with butyrate, some cytotoxicity was detected in these cells with baculovirus at MOI 1000. Similar results were obtained in early Wattanabe hereditary hyperlipidemia (WHHL) rabbit fibroblasts, except that no cytotoxic effects on baculovirus were detected in the presence of butyrate at MOI 1000 (data not shown).

In vivo gene transfer

To determine if baculovirus can be used in in vivo gene transfer, NZW rabbits were killed 3,7 and 14 days after gene transfer. Due to nuclear targeting of β-galactosidase expression, dark X-Gal staining was located in the nuclear cells of the transduced cells. The number of cells positive for β-galactosidase activity was calculated from the baculovirus-transduced (1 × ^{10 9} pfu per artery) and compared to similarly treated adenovirus-transduced arteries. Both viruses modulated the delivery of marker genes to the vessel wall. On day 3 the number of transgene positive cells for baculovirus- and adenovirus-treated arteries was 12 ± 5 and 23 ± 7 β-galactosidase positive cells / mm 2 (11 ± 4 and 19) of the outer membrane, respectively. ± 6 mm 2 of all artery walls). On day 7, the corresponding values were 17 ± 6 and 22 ± 7 (15 ± 5 and 18 ± 6). On day 14, the corresponding values were 0.1 ± 0 and 0.2 ± 0.1 (0.1 ± 0 and 0.1 ± 0.1). Therefore, baculovirus-regulated gene expression was transient with similar efficiencies and time patterns as for adenovirus-regulated gene transfer. Transgene expression in the arterial wall was also demonstrated by RT-PCR.

The mean value of the endometrial / membrane ratio (all arteries) was 0.18 ± 0.03 in baculovirus-treated arteries and 0.12 ± 0.01 in adenovirus-treated arteries, indicating that the process did not damage the vascular wall. At 7 days after gene transfer, histology of the arteries did not detect any β-galactosidase activity outside of the outer membrane. In both viruses, macrophages infiltrated and certain T cells were detected individually in arteries transduced by RAM-11 and MCA-805 immunostaining, respectively. Artery structure and endothelial remained intact throughout the experiment. Histological findings at all time points are summarized in Table 3.

Transduction of RAASMC and ECV-304 Cell Lines with baculovirus and adenovirus Cell line MOI Butyrate (mM) Positive cell (%) Baculovirus Adenovirus RAASMC 200 0 1.0 ± 0.3 1.7 ± 0.8 10 52.5 ± 0.8 42.1 ± 1.1 1000 0 5.3 ± 1.3 48.0 ± 5.8 10 90.6 ± 1.6 87.6 ± 2.3 ECV-304 200 0 0 44.2 ± 2.9 10 2.6 ± 1.7 89.4 ± 1.9 1000 0 0.3 ± 0.3 87.51 ± 4.0 10 20.8 ± 7.7 97.6 ± 1.4

± s.e.m.

MTT assessment for cytotoxicity of baculovirus and adenovirus in RAASMC cells MOI Butyrate (mM) Baculovirus (%) ^* Adenovirus (%) ^* 200 0 82 ± 3 98 ± 4 10 78 ± 2 101 ± 2 1000 0 81 ± 1 99 ± 1 10 65 ± 1 90 ± 3

^* Percent survival of RAASMC cells compared to mock-transduced cells

± s.e.m.

Summary of Histological Findings in Arteries Transduced 3,7, and 14 Days After Gene Transfer Viewpoint virus Macrophage PMN cells Endothelial contact 3 days Adenovirus ++ + Yes Baculovirus ++ ++ Yes 7 days Adenovirus + + Yes Baculovirus + + Yes 14 days Adenovirus ++ - Yes Baculovirus ++ - Yes

-, As compared to the control artery; + Light; ++ Medium and +++ extreme penetration.

PMN, Polymorphonucle

Example 2

Essentially using the same procedure as in Example 1, it was shown that baculovirus gene transfer also occurs in the brain and skeletal muscle. Using baculovirus / lacZ, the rabbit brain exhibits positive transduction in various types of brain cells, especially chorionic total cells and endothelial cells in the ventricles. The profile of the transfected cells is clearly different from that of the adenovirus.

In addition, transfection of baculovirus has been demonstrated in rabbit skeletal muscle. The baculovirus (1.8 × ^{10 10} PFU) encoding lacZ was injected directly into the adrenal muscle of NZW rabbits via a 25 G needle. Infection volume is 0.5 ml. Tissue samples were collected 7 days after gene transfer and X-Gal staining was performed overnight. These results clearly indicate that baculovirus can be used for transfection of ie arterial cells as well as various cell types in mammals.

The accompanying figures illustrate the construction of nuclear-targeting β-galactosidase-encoding baculovirus transfection cassettes. In principle, this is a standard open domain baculovirus with a polyhedrin promoter, which is cloning the restriction region and the CMV-NT lacZ expression cassette. The lacZ expression cassette is opposite to the polyhedrin promoter. CMV-nt lacZ expression cassette is SEQ ID NO; 7.

Claims (11)

The gene is provided to a baculovirus vector, wherein the vector is applied to a body compartment with little or no blood. The method of claim 1, wherein said delivery is perivascular. The method of claim 1 or 2, wherein said gene product is an agent of the VEGF receptor. The method of claim 1 for treating aberrant proliferation of the artery. The method of claim 1 or 2, wherein said compartment comprises arterial cells. The method of claim 1 or 2, wherein the compartment comprises brain cells. The method of claim 1 or 2, wherein said compartment comprises skeletal muscle. A device for perivascular delivery of a gene product, wherein the gene is provided to a baculovirus vector in which the gene can be expressed. The device of claim 8 in the form of a collar. The device of claim 8 in the form of a wrap. The device of claim 8, wherein the gene product is an agonist of a VEGF receptor.