NO20025657L - Use of Baculovirus Vectors in Gene Therapy - Google Patents
Use of Baculovirus Vectors in Gene Therapy Download PDFInfo
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- NO20025657L NO20025657L NO20025657A NO20025657A NO20025657L NO 20025657 L NO20025657 L NO 20025657L NO 20025657 A NO20025657 A NO 20025657A NO 20025657 A NO20025657 A NO 20025657A NO 20025657 L NO20025657 L NO 20025657L
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- cells
- baculovirus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Description
Oppfinnelsens områdeField of the invention
Foreliggende oppfinnelse angår genlevering ved anvendelse av en viral vektor. The present invention relates to gene delivery using a viral vector.
Bakgrunn for oppfinnelsenBackground for the invention
Effektiv genoverføring ville være et fordelaktig verktøy for behandling av vaskulære sykdommer, så som post-angioplasti restenose, post-bypass aterosklerose, perifer aterosklerotisk sykdom, stenose av vaskulære protese-anastomoser og trombedannelse. Forskjellige teknikker er utviklet for dette formål; se for eksempel Yla-Herttuala et al, J. Clin. Invest. 95:2692-8 (1995) og Laitinen et al, Hum. Gene. Ther. 8:1645-50 (1997). 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 beskriver en periadventitial krave som kan anvendes for arteriell genoverføring under vaskulær kirurgi. Imidlertid er det et kontinuerlig behov for enklere og mer effektive genoverføringsvektorer. Bare en temporær ekspresjon av transgenet kan være nødvendig for å oppnå en fordelaktig biologisk effekt ved kardiovaskulære applikasjoner; se Yla-Herttuala et al, Lancet 355:213-222 (2000). 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).
Baculovirus har lenge vært anvendt som biopesticider og som verktøy for effektiv rekombinant proteinproduksjon i insektceller. De er generelt betraktet som sikre, på grunn av den naturlige høye artsspesifisitet og fordi de ikke er kjent å propagere hos noen ikke-virvelløs vert. Selv om virionene er vist å entre visse cellelinjer avledet fra virveldyrarter, er det ikke noe bevis på at viral genekspresjon har blitt detektert ved anvendelse av naturlige virus. Imidlertid kan Autographa californica multippelt nukleært polyhedrose-virus (AcMNPV), inneholdende en passende eukariot promoter, overføre og uttrykke målgener effektivt i mange pattedyrcelletyper; se for eksempel Hofmann et al, PNAS USA 92:10099-10103 (1995). I tillegg har Barsoum et al, Hum. Gen. Ther. 8:2011-8 (1997) angitt at baculovirus som har det vesikulære stomatitt-virus C glykoprotein i dets kappe, betydelig øker effektiviteten av transduksjon av humane hepatom-cellelinjer og utvider området av pattedyrcelletyper som kan transduseres av baculovirus. Stabil transduksjon av pattedyrceller av baculovirus er oppnådd ved enten å inkludere en ekspresjonskassett som koder for en dominant selekterbar markør i baculovirus-genom eller ved å anvende hybrid baculovirus-adeno-assosiert virusvektor; se Condreay et al, PNAS USA 96:127-132 (1999) og Palombo et al, J. Virol. 72:5025-34 (1998). 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) rapporterte mislykkede forsøk på å anvende baculovirus for in vivo genlevering til mus og rotter ved systemisk eller intraportal anvendelse så vel som ved direkte injeksjon i lever-parenkym. Én grunn for dette er antagelig inaktivering av baculovirus av den klassiske bane av serum-komplementsystem. 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 beskriver baculovirus-vektorer og anvendelse av dem for genoverføring til nerveceller i virveldyr. WO-A-00/05394 describes baculovirus vectors and their use for gene transfer into vertebrate nerve cells.
Oppsummering av oppfinnelsenSummary of the invention
Det er nå funnet at inaktivering av baculovirus kan unngås. Spesielt er det vist at baculovirus kan mediere periadventitial genoverføring til kanin-karotidarterier med en effektivitet sammenlignbar med adenovirus. Letthet ved manipulering og rask konstruksjon av rekombinante baculovirus, deres mangel på cytotoksisitet i pattedyrceller selv ved en høy infeksjonsmultiplisitet, deres naturlige manglende evne til å replikere i pattedyrceller og deres store kapasitet for insersjon av fremmede sekvenser, gjør baculovirus til et meget egnet verktøy for in vivo genterapi. 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.
Foreliggende oppfinnelse kan utnytte de fordelaktige egenskapene til baculovirus, i en egnet vektor fra hvilken genet blir uttrykt, hvis administrert ( in eller ex vivo) til et sted på kroppen hvor det ikke er noe blod eller som er i det vesentlige fritt for blod. Således tillater periadventitial eller, mer spesifikt, krage-mediert lokal genlevering genoverføring i det vesentlige i fravær av serum, for således å unngå skadelige effekter av serumkomponenter. Den nye metoden unngår også to andre hovedproblemer som møtes ved systemisk genlevering, dvs. en rask redistribusjon av viruset fra injeksjonsstedet og et fall i den lokale konsentrasjon av viruset. 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.
Beskrivelse av foreliggende oppfinnelseDescription of the present invention
Egnede leveringssystemer, aktive materialer, preparater, doser etc, er illustrert i WO-A-98/20027 og også WO-A-99/55315 (hvis innhold inntas her ved referanse). Således kan, bare som eksempel, leveringskonstituenten være en krave eller et omslag. Sammenlignet med publikasjonene er vektoren for genlevering et baculovirus. 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.
Baculovirus er selvfølgelig kjent og fagfolk vil være i stand til å konstruere en hvilken som helst egnet vektor for anvendelse ved foreliggende oppfinnelse. Det vil også være åpenbart at den brede kunnskap om baculovirusbiologi og AcMNPV-genom vil hjelpe konstruksjon av forbedrede andregenerasjons-virus for genoverføringsanvendelser. Enkelhet av konstruksjon og kapasitet til å akseptere store fremmede DNA-fragmenter (>20 kbp), tillater utvikling av baculovirus som har forbedret eller målrettet celle-tropisme sammen med mer stabil, temporal og celletype-spesifikk kontroll av transgen ekspresjon. Et rekombinant baculovirus for anvendelse ved oppfinnelsen kan formuleres i et medikament for terapeutisk anvendelse på kjent måte. 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.
Ruter og steder for administrering av oppfinnelsen omfatter intraokulær anvendelse, intraartikulær anvendelse, overflate intradermal anvendelse, urinledere, blære, eggleder, galleblære, ryggmarg, 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,
cerebrospinalvæskekammer, plevrahulen og intraperitonealhulen. Steder som har vært anvendt (se eksemplene) er arterier, hjerne og skjelettmuskel, omfattende, eksempelvis, mycocytter, satellittceller og regenererende myoblaster. Genlevering kan utføres ved direkte injeksjon eller ved forskjellige typer katetere. 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.
Hvis hensiktsmessig kan kroppsdeler gjøres "blodløse" under kirurgi. Denne teknikk blir ofte anvendt ved ben- eller arm-kirurgi ved å stramme tett trykk rundt arm eller lår, for således å forhindre blodstrøm. Kroppsdelen kan deretter perfuseres med saltvann for å fjerne blod, og baculovirus-transfeksjon kan deretter utføres. 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.
Oppfinnelsen kan anvendes for levering av en agonist for en VEGF-reseptor, f.eks. beskrevet mer detaljert i WO-A-98/20027. Videre, ved egnet valg av gen, kan den anvendes ved behandling av kreft, f.eks. i hjernen. 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.
Et ytterligere aspekt ved foreliggende oppfinnelse angår transplantat-organer og -kar som kan perfuseres med saltvann ex vivo og underkastes ex vivo baculovirus-injeksjon. 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.
Det følgende eksperimentelle arbeid illustrerer oppfinnelsen. The following experimental work illustrates the invention.
Eksempel 1Example 1
Fremstilling av rekombinante baculovirusProduction of recombinant baculoviruses
Virus ble konstruert ved anvendelse av overføringsvektoren pFASTBad (pFB) (Gibco BRL, Life Technologies, Gaithersburg, MD, USA). En nukleær målrettet/3-galaktosidase- ((3nt-Gal) kassett med en cytomegalovirus- (CMV) promoter ble innsatt i Sful-setet av pFB i revers orientering med hensyn til polyhedrin-promoteren, for å danne plasmidet pFBCMV-j3nt- 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-
Rekombinant virus ble dannet ved anvendelse av et BacTo-Bac™ baculovirus-ekspresjonssystem (Gibco BRL). Virus ble amplifisert i Spodoptera frugiperda 9- (Sf9) suspensjonskulturer (SF-900 medium, Gibco BRL) i 3 dager ved anvendelse av celledensitet på 2 x 10<6>celler/ml. For 50 ml kultur ble 200 ( i\ av primær transfeksjons-supernatant anvendt som inokulum. For å oppnå 1 liter av amplifisert virus ble 2 ml amplifisert virus-lager anvendt som inokulum. Celledyrkningsmediet ble sentrifugert ved 16.000 g i 20 min. ved romtemperatur for å fjerne cellerester. Den klarede supernatant ble overført til ultrasentrifugerør underlagt med 1,5 ml 25% sukrose i fosfatbufret saltvann (PBS) og virus ble konsentrert ved sentrifugering (120.000 g. 4EC, 1,51). Viruspellet ble resuspendert i 35 ml iskald PBS, overført til ultrasentrifugerør inneholdende 3 ml 25% sukrose i PBS og sentrifugert som ovenfor. Endelig viruspellet ble resuspendert i 10 ml kald PBS, filtrert gjennom 0,45 ixm filter og holdt ved 4EC beskyttet fra lys for videre anvendelse. Virustiter ble bestemt ved et plaque-forsøk på Sf9-celler. Viruspreparatene ble analysert for lipopolysakkarid og bakteriologiske forurensninger. 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.
Fremstilling av rekombinante adenovirusProduction of recombinant adenoviruses
Nukleær-målrettet LacZ som koder for adenovirus (pCMVnls/acZAd5) ble konstruert og fremstilt som beskrevet av Laitinen et al, supra. Virus-preparater ble analysert for replikasjonskompetente virus, lipopolysakkarid og bakteriologiske forurensninger som beskrevet av Laitinen et al, Hum. Gen Ther. 9:1481-6(1998). 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).
Genoverføring in vitroGene transfer in vitro
Kanin-aortaceller (RAASMC; Yla-Herttuala et al (1995), supra) og humane karsinom/endotel-celle-lignende ECV-304-celler (ATTC CRL-1998) ble platet ut med en densitet på 10.000 celler pr. brønn (Falcon Culture Slide, Becton Dickinson, Meylan, Frankrike). Celler fikk binde i 3 timer før transduksjon i serumfritt medium (DMEM, 100 enheter/ml penicillin og 100 jig/ml streptomycin, Gibco BRL). Virus ble satt til medium med MOI på 200 eller 1000 og celler ble inkubert i 90 min. ved 37EC. Etter transduksjon'ble vekstmedium inneholdende 10% føtalt bovint serum tilsatt enten med eller uten 10 mM n-smørsyre (Sigma, St. Louis, MD, USA). Etter 18 timers inkubering ble mediene fjernet og cellene ble vasket tre ganger med PBS. Cellene ble fiksert med 1,25% glutaraldehyd i 15 min. og vasket tre ganger med PBS. X-Gal (MBI Fermentas, Litauen) merkingsløsning (1 mg/ml, 2 mM MgCI2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 x PBS) ble satt til cellene og inkubert i 3 timer ved 37EC. Cellene ble deretter vasket med PBS og videre fiksert med 4% paraformaldehyd (PFA) i 10 min. Etter vasking med PBS ble cellene motmerket med Mayers Carmalum i 5 min. Blue X-Gal-positive celler ble tellet og transduksjonseffektiviteten ble uttrykt som prosentdelen av positive celler av det totale antall av celler. 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- forsøkONPG trial
/acZ-kodet /?-galaktosidase-aktivitet i transdusert RAASMC-celler ble målt ved anvendelse av et kolorimetrisk substrat, o-nitrofenyl-/3-D-galaktopyranosid (ONPG, Sigma), som beskrevet av Ruponen et al, Biochim. Biophys. Acta 1415:331-41 (1999). /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 toksisitetsforsøkIn vitro toxicity tests
Celler ble platet ut i 96-brønn plater med en densitet på 20.000 celler pr. brønn i 100/il vekstmedium bestående av DMEM med 10% føtalt bovint serum og antibiotika (100 enheter/ml penicillin og 100/xg/ml streptomycin). Virus-transduksjoner ble utført som for transduksjons-effektivitetsforsøket og celler ble inkubert i 48 timer ved 37EC. Vekstmedium ble fjernet og celler ble vasket med PBS. Serum-fri DMEM uten fenolrød, inneholdende MTT-løsning (3-(4,5- dimetyltiazolyl-2)-2,5-difenyl-tetrazoliumbromid, 5 mg/ml, endelig konsentrasjon 0,3 mg/mi) ble deretter tilsatt og cellene ble inkubert i 2 timer. For å oppløse de mørkeblå formazankrystaller ble MTT-løsning fjernet, 150 /il av 1 M DMSO ble satt til hver brønn og blandet grundig. Absorbans blejnålt ved 570 nm. Overlevelse-prosentdel ble beregnet sammenlignet med absorbans av ikke-virus- eller ikke-butyrat-brønner (100% overlevelse). 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).
DyreforsøkAnimal testing
New Zealand hvite (NZW) hannkaniner [ n = 12; 2,8-3,7 kg) ble anvendt. Fentanyl-fluanison (0,3 ml/kg, s.c; Janssen Pharmaceutica, Beerse, Belgia) og midazolam (1,5 mg/kg, i.m., Roche, Basel, Sveits) ble anvendt for anestesi. Venstre og høyre karotid-arterier ble eksponert ved anvendelse av midtlinje hals-snitt. Arterien ble forsiktig separert fra det omgivende vev og en 3 cm lang mykplast "silastic" krave (se Laitinen et al (1997), supra) ble plassert rundt den. Kaninene ble bedøvet igjen for genoverføring, som ble utført 5 dager etter installasjon av kraven på nøyaktig samme måte som beskrevet av Laitinen et al (1997), supra, som sammenlignet transfeksjonseffektivitet av plasmid/liposomer, pseudotypet retrovirus og adenovirus. Kravene ble åpnet og fylt med 500/il av genoverføringsløsningen inneholdende 1 x 10<9>pfu av adenovirus eller baculovirus. For hvert dyr ble venstre karotid-arterie anvendt for adenovirus- og høyre karotid-arterie for baculovirus-behandling. Fire kaniner ble avlivet 3, 7 og 14 dager etter genoverføringen og arteriene ble fjernet for histologiske analyser. Alle dyreprosedyrer var godkjente. 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.
Histologisk analyseHistological analysis
Arterier med krave ble delt i tre like deler: den proksimale tredjedel ble immersjons-fiksert i 4% PFA/15% sukrose (pH 7,4) i 4 timer, skyllet i 15% sukrose (pH 7,4) natten over og innleiret i paraffin. Den mediale del ble immersjons-fiksert i 4% PFA/PBS (pH 7,4) i 30 min, skyllet i PBS (pH 7,2) og innleiret i OCT-forbindelse (Miles Scientific, Naperville, IL, USA). Den distale del ble hurtigfrosset i flytende nitrogen og lagret ved -70EC for mRNA-isolering og revers transkriptase-polymerasekjedereaksjon (RT-PCR). 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).
Tilfeldig valgte frosne snitt (10/tm) fra hver kanin ble merket med X-Gal (MBI Fermentas) i 18 timer for å identifisere Ø-galaktosidase-positive celler. Genoverføringseffektivitet ble beregnet som X-Gal-positive celler pr. mm<2>av adventitia eller hel arterievegg i åtte tilfeldig valgte snitt av to uavhengige observatører. Gjennomsnittsverdier V standard middel avvik (SEM) for resultatene er angitt. Paraffin-snitt ble anvendt for immunocytokjemisk deteksjon av endotel (CD-31; 1:50 fortynning; Dako, Hamburg, Tyskland), makrofager (RAM-11; 1:100 fortynning; Dako), glattmuskelceller (HHF-35; 1:50 fortynning; Dako) og T-celler (MCA-805; 1:100 fortynning; Dako) som beskrevet av Yla-Herttuala (1995), supra. Kontroller for immunomerkingene omfattet snitt hvor det første antistoff ble utelatt og snitt inkubert med klasse-og arts-matchede immunoglobuliner. Morfometri og bildeanalyse ble utført ved anvendelse av hematoxylin-eosin-merkede paraffin-snitt og Image-Pro Plus™ programvare med Olympus AX70 mikroskop (Olympus Optical, Japan). 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- PCRRT-PCR
Total RNA ble ekstrahert fra transduserte karotidarterie-segmenter ved anvendelse av Trizol-reagens (Gibco-BRL) og behandlet med overskudd av RQ1 RNAse-fri DNAse (Promega, Madison, Wl, USA). M-MulV revers transkriptase (MBI Fermentas) ble anvendt for cDNA-syntese. Primere (20 pmol hver) for /acZ ble utformet for å skille mellom endogene og transduserte gener ved seleksjon av 5'-primerene fra CMV-promoter og 3'-primerene fra den kodende region. Dynazyme-polymerase (Finnzymes, Esbo, Finland) ble anvendt for amplifisering. 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-amplifisering var primerene SEKV ID NR:1 for adenovirus (A) og SEKV ID NR:2 for baculovirus (B) som forover primere og SEKV ID NR:3 for begge som en revers primer. Varm start (95EC 5 min; 58EC 3 min) ble fulgt av 39 cykler, hver bestående av 95EC 1 min, 58EC 2 min, 72EC 3 min med den endelige utvidelse 10 min ved 72EC. 5/il av det første PCR-produkt ble anvendt for den andre PCR med forover primere SEKV ID NR:4 (A) og SEKV ID NR:5 (B). Den reverse primer for begge var SEKV ID NR:6. Den første PCR-cyklus var 95EC i 5 min. fulgt av 20 cykler som i den første PCR. 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.
Genoverføring in vitroGene transfer in vitro
For å teste baculovirus-lager ble RAASMC- og ECV-304-celler transdusert med en infeksjonsmultiplisitet (MOI) på 200 eller 1000 pfu pr. celle i fravær eller nærvær av 10 mM natriumbutyrat og prosentdelen av X-Gal-positive celler ble tellet. Resultatene ble sammenlignet med celler transdusert med /acZ-adenovirus under identiske betingelser (Tabell 1). I overensstemmelse med publiserte resultater, (Condreay ef al, supra) øket tilsetning av butyrat til cellekulturer bemerkelsesverdig ekspresjonen av transgenet, spesielt med baculovirus. RAASMC-cellene syntes å være mer mottagelige for baculovirus-transduksjon (91% infisert med MOI 1000) enn ECV-304-celler (21% infisert med MOI 1000). Nivåer av /2-galaktosidase-aktivitet i RAASMC-cellene ble også målt ved et kvantitativt biokjemisk forsøk med o-nitrofenyl-/3-D-galaktopyranosid (ONPG). Resultatene var på linje med X-Gal-merking som viser en økning i transgenekspresjonen etter butyrat-behandling i de baculovirus-transduserte celler. 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 toksisitetIn vitro toxicity
3-(4,5-dimetyltiazol-2-yl)-2,5-difenyltetrazoliumbromid- (MTT) forsøk ble anvendt for å måle cytotoksisiteten av viruspreparatene (Tabell 2). Hverken baculovirus eller adenovirus viste noen vesentlig cytotoksisitet for RAASMC-celler i fravær av butyrat ved MOI 200 eller 1000. Sammen med butyrat ble imidlertid noe cytotoksisitet detektert i disse celler med baculovirus ved en MOI på 1000. Lignende resultater ble oppnådd med primær WHHL- (Watanabe arvelige hyperlipidemiske) kanin-fibroblaster bortsett fra at ingen cytotoksiske effekter ble detektert for baculovirus i nærvær av butyrat ved en MOI på 1000 (data ikke vist). 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).
Genoverføring in vivoGene transfer in vivo
For å bestemme hvorvidt baculovirus kan anvendes for in vivo genoverføring, ble NZW-kaniner avlivet 3, 7 og 14 dager etter genoverføring. På grunn av nukleær målretting av /3-galaktosidase-ekspresjon, ble intens X-Gal-merking lokalisert i kjernene til de transduserte celler. Antallet celler positive for/3-galaktosidase-aktivitet ble beregnet fra baculovirus-transduserte 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 pr. arterie) karotid-arterier og ble sammenlignet med tilsvarende behandlede adenovirus-transduserte arterier. Begge virus medierte levering av markørgenet til karveggen. Antallet transgene positive celler for baculovirus- og adenovirus-behandlede arterier på dag 3 var henholdsvis 12 V 5 og 23 V 7/3-galaktosidase-positive celler/mm<2>adventitia (11 V 4 og 19 V 6/mm<2>av hele arterieveggen). På dag 7 var de tilsvarende verdier 17 V 6 og (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 og 18 V 6). På dag 14 var verdiene 0,1 V 0 og 0,2 V 0,1 (0,1 V 0 og 0,1 V 0,1). Baculovirus-mediert genekspresjon var således transient med en lignende effektivitet og tidsmønster som den til adenovirus-mediert genoverføring. Transgen ekspresjon i arterieveggen ble også verifisert med 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.RT-PCR.
Den gjennomsnittlige verdien av intima/media-forhold (alle arterier) var 0,18 V 0,03 for baculovirus- og 0,12 V 0,01 for adenovirus-behandlede arterier, som indikerer at metodene ikke skader karveggen. Histologi for arterier 7 dager etter genoverføring, detekterte ingen/3-galaktosidase-aktivitet utenfor adventitia. For begge virus ble makrofag-infiltrater og noen T-celler detektert i de transduserte arterier ved henholdsvis RAM-11- og MCA-805-immunomerking. Den arterielle struktur og endotelet forble intakt gjennom alle forsøkene. Histologiske funn ved alle tidspunkter er oppsummert i Tabell 3. 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.
Eksempel 2 Example 2
Ved anvendelse av i det vesentlige samme prosedyre som i Eksempel 1, er det vist at baculovirus-genoverføring virker også på hjerne og skjelettmuskel. Ved anvendelse av baculovirus//acZ viser rottehjeme positiv transfeksjon i forskjellige typer av hjerneceller, spesielt i choroid plexus-celler i ventrikler og endotelceller. Profilen til transfekterte celler er klart forskjellig fra den til adenovirus. 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.
Videre er baculovirus-transfeksjon demonstrert i kanin-skjelettmuskel. Baculovirus som koder for lacZ (1,8 x 10<10>PFU) ble direkte injisert i adduktor-muskel til NZW-kanin via en 25 G nål. Injeksjonsvolumet var 0,5 ml. Vevprøver ble oppsamlet 7 dager etter genoverføringen og X-Gal-merking ble utført natten over. Disse resultater indikerer klart at baculovirus kan anvendes for transfeksjon av mange celletyper hos pattedyr, dvs. ikke bare arterielle celler. 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.
Den medfølgende tegningen illustrerer konstruksjonen av en nukleær-målrettet/3-galaktosidase-kodende baculovirus-transfeksjonskasett. I The accompanying drawing illustrates the construction of a nuclear-targeting/3-galactosidase-encoding baculovirus transfection cassette. IN
prinsippet er dette et standard ålment baculovirus med polyhedrin-promoter, i hvilket er klonet restriksjonsseter og CMV-NT /acZ-ekspresjonskassett. /acZ-ekspresjonskassett er orientert motsatt polyhedrin-promoteren. Sekvensen av CMV-nt /acZ-ekspresjonskassetten er i SEKV ID NR:7. 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.
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