EP1392837A2 - Gentransfer mittels eines baculoviralen vektors - Google Patents

Gentransfer mittels eines baculoviralen vektors

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Publication number
EP1392837A2
EP1392837A2 EP02730459A EP02730459A EP1392837A2 EP 1392837 A2 EP1392837 A2 EP 1392837A2 EP 02730459 A EP02730459 A EP 02730459A EP 02730459 A EP02730459 A EP 02730459A EP 1392837 A2 EP1392837 A2 EP 1392837A2
Authority
EP
European Patent Office
Prior art keywords
gene
baculovirus
cells
baculoviruses
brain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02730459A
Other languages
English (en)
French (fr)
Inventor
Seppo A.I. Virtanen Institute YLA-HERTTUALA
Kari Juhani A.I. Virtanen Institute AIRENNE
Pauliina A.I. Virtanen Institute LEHTOLAINEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ark Therapeutics Ltd
Original Assignee
Ark Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/GB2001/002383 external-priority patent/WO2001090390A1/en
Priority claimed from GBGB0128620.2A external-priority patent/GB0128620D0/en
Application filed by Ark Therapeutics Ltd filed Critical Ark Therapeutics Ltd
Publication of EP1392837A2 publication Critical patent/EP1392837A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • This invention relates to gene delivery using a viral vector.
  • 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 prosthesis anastomoses, and thrombus formation.
  • vascular diseases such as post-angioplasty restenosis, post-bypass atherosclerosis, peripheral atherosclerotic disease, stenosis of vascular prosthesis anastomoses, and thrombus formation.
  • vascular diseases such as post-angioplasty restenosis, post-bypass atherosclerosis, peripheral atherosclerotic disease, stenosis of vascular prosthesis 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 La
  • WO-A-98/20027 discloses a periadventitial collar that can be used for arterial gene transfer during vascular surgery.
  • WO-A-98/20027 discloses a periadventitial collar that can be used for arterial gene transfer during vascular surgery.
  • Baculoviruses have long been used as biopesticides and as tools for efficient recombinant protein production in insect cells. They are generally regarded as safe, due to the naturally 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, no evidence of viral gene expression has been detected using natural viruses. However, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), containing an appropriate eukaryotic promoter, is able to transfer and express target genes efficiently in several mammalian cell types; see, for example, Hofmann et al, PNAS USA 92:10099-10103 (1995).
  • AcMNPV Autographa californica multiple nuclear polyhedrosis virus
  • baculovirus having the vesicular stomatitis virus C glycoprotein in its envelope significantly increases the efficiency of transduction of human hepatoma cell lines and broadens the range of mammalian cell types that can be transduced by baculoviruses.
  • Stable transduction of mammalian cells by baculoviruses has been achieved by either including an expression cassette encoding a dominant selectable marker into baculovirus genome or by using hybrid baculovirus-adeno-associated virus vector; see Condreay etal, PNAS USA 96:127-132 (1999), and Palombo etal, J. Virol. 72:5025-34 (1998).
  • WO-A-00/05394 discloses baculovirus vectors and their use for gene transfer to the nerve cells of vertebrates. Summary of the Invention
  • baculoviruses are able to mediate periadventitial gene transfer to rabbit carotid arteries with an efficiency comparable to adenoviruses.
  • This invention is able to use the advantageous properties of baculoviruses, in a suitable vector, from which the gene is expressed, if administered (in or ex vivo) to a body site at which there is no blood, or which is essentially free of blood.
  • baculoviruses in a suitable vector, from which the gene is expressed, if administered (in or ex vivo) to a body site at which there is no blood, or which is essentially free of blood.
  • collar-mediated local gene delivery allows gene transfer essentially in the absence of serum, thus avoiding deleterious effects of serum components.
  • the novel method also avoids two other major 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.
  • baculoviruses specifically transduced cuboid epithelium of the choroid plexus in ventricles and that the transduction efficiency was as high as 76% ⁇ 14, whereas adenoviruses showed preference to corpus callosum glial cells and ventricular ependymal lining. Only a modest microglia response was seen after the baculovirus transduction, whereas the adenovirus gene transfer led to a strong microglia response. Sensitive nested RT-PCR revealed transgene expression in hindbrain and in ectopic organs including spleen, heart and lung, which indicates that some escape of both vectors occur to ectopic organs after local gene transfer to brain.
  • baculovirus vectors can be used for local intracerebral gene therapy.
  • the knowledge of the cell type specificity of the vectors offers a possibility to achieve targeted gene delivery to distinct brain areas.
  • Baculoviruses seem to be especially useful for the targeting of choroid plexus cells. Since choroid plexus cells are involved in the production of cerebrospinal fluid, they are a target for the production of secreted therapeutic proteins in the brain. It may be deduced that, by utilizing the naturally restricted cell tropism, baculoviruses provide an efficient tool for gene delivery to cerebral choroid plexus cells and may become useful for gene therapy of several types of brain disorders. Description of the Invention
  • Suitable delivery systems, active materials, formulations, dosages etc, are illustrated in WO-A-98/20027 and also WO-A-99/55315 (the contents of which are incorporated herein by reference).
  • the delivery vehicle may be a collar or wrap.
  • the vector for gene delivery is a baculovirus.
  • Baculoviruses are of course known, and the skilled person will be able to construct any suitable vector for use in this invention. It will also be evident that the broad knowledge of baculovirus biology and Ac NPV genome will aid engineering of the improved second-generation viruses for gene transfer applications. The ease of construction, and capacity to accept large foreign DNA-fragments (>20 kbp), allows the development of baculoviruses having enlarged 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 may be formulated into a medicament for therapeutic use, in known manner.
  • Routes and sites of administration, for the invention include intra-ocular application, intra-articular application, superficial intra-dermal application, ureters, bladder, Fallopian tubes, gall bladder, spinal cord, cerebrospinal fluid compartment, pleural cavity and intraperitoneal cavity.
  • Sites that have been used are arteries, brain and skeletal muscle, including, by way of example, mycocytes, satellite cells and regenerating myoblasts.
  • Gene delivery may be done via direct injection or various types of catheters.
  • body parts can be made "bloodless” during surgery. This technique is often used in leg or arm surgery by putting tight pressure around arm or thigh, thus preventing blood flow. The body part may then be perfused with saline to remove blood, and baculovirus transfection can then be done.
  • the invention can be used for the delivery of an agonist of a VEGF receptor, e.g. described in more detail in WO-A-98/20027.
  • a VEGF receptor e.g. described in more detail in WO-A-98/20027.
  • the gene that is delivered may be designed for enzyme replacement therapy.
  • the active agent may cause the production of NO, e.g. to treat subarachnoid hemorrhage; a suitable gene is for endothelial NO synthase. More generally, the active agent may be for any condition that affects or can be mediated via the spinal cord/CNS.
  • a further aspect of the invention relates to transplant organs and vessels which can be perfused with saline ex vivo and subjected to ex vivo baculovirus injection.
  • Example shows that baculovirus gene transfer works in brain and skeletal muscle.
  • rat brain shows positive transfection in various 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 adenoviruses.
  • 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 rabbit 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 staining was performed overnight.
  • the accompanying drawing illustrates the construction of a nuclear-targeted ⁇ -galactosidase-encoding baculovirus transfection cassette.
  • this is a standard public domain baculovirus with polyhedrin promoter, into which have been cloned restriction sites and the CMV-NT lacZ expression cassette.
  • the lacZ expression cassette is oriented opposite to the polyhedrin promoter.
  • the sequence of the CMV-nt lacZ expression cassette is in SEQ ID NO:7.
  • + 1 corresponds to the transcriptional start for the polyhedrin promoter.
  • the ATG was mutated to an ATT.
  • Rats 200-250 g were anesthetized intraperitoneally with a solution (0.150 ml/100 g) containing fentanyl-fluanisone (Janssen-Cilag, Hypnorm®, Buckinghamshire, UK) and midazolame (Roche, Dormicum®, Espoo Finland), placed into stereotaxic apparatus (Kopf Instruments) and 20 ⁇ l of the virus in PBS/0.1% sucrose was injected during 2x10 min periods using Hamilton syringe with a 27-gauge needle. Procedure was repeated in three consecutive days.
  • Rats received 10 8 plaque forming units (pfu) of both vectors.
  • Rats were perfused with 1x PBS by transcardiac route for 10 min followed by fixation with 4% paraformaldehyde/0.15 sodium-phosphate buffer(pH 7.4) for 10 min.
  • Brain was removed and divided at the injection site into two coronal pieces. Samples from fore and hindbrain, liver, kidney, heart, spleen, lung and skeletal muscle (psoas majo ⁇ were taken. Tissue samples were rinsed in 1 x PBS and embedded in O.C.T. compound (Tissue-Tek, Sakura) or snap-frozen for nested PCR analysis.
  • the LacZ activity of the sections was analysed with 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside (X-Gal; MBI Fermentas) for 18 h to identify ⁇ -galactosidase positive cells.
  • Gene transfer efficiency was calculated from 5-8 randomly selected sections at the injection site from each animal as a percent of the ⁇ -galactosidase positive nuclei of the total number of nuclei in the specific cell types (i.e. choroid plexus, ependyma, corpus callosum) from the area of 200 x 200 ⁇ m.
  • Monoclonal antibodies CD31 (1 :200, Dako), anti-fibrillary acidic protein (GFAP 1:400, Boehringer Mannheim) and CD11b (OX-421 :200, Serotec) were used to identify endothelial, astrocytic and microglial cells, respectively.
  • Avidin-biotin-HRP system and biotinylated secondary antibodies with DAP staining were used for signal detection (Vector Elite, Vector Laboratories, Burlingam, California). Sections were counterstained with Mayer's Carmalum or hematoxylin and data were collected with Image-Pro Plus software with Olympus AX70 microscope (Olympus Optical, Japan).
  • RNA samples for immunostaining included incubations with class- and species-matched immunoglobulins and incubations without primary antibodies.
  • RT-PCR Total RNA from spleen, liver, kidney, lung, heart, skeletal muscle and transduced brain samples was extracted using TRIZOL reagent (Gibco-BRL). Samples were subsequently treated with RQ1 RNasefree DNase (Promega, Madison, Wl, USA) to eliminate DNA contamination. M-MuLV reverse transcriptase (MBI Fermentas) was used for cDNA synthesis. The RT-PCR protocol is described above. Dynazyme DNA polymerase (Finnzymes, Espoo, Finland) was used to amplify cDNA template.
  • Primer (20 pM/reaction) sequences for LacZ gene were SEQ ID NO. 1 for adenovirus and SE ID NO. 2 for baculovirus as forward primers, and SEQ ID NO. 3 for both viruses as a reverse primer.
  • 39 cycles with 1 min denaturation (95°C), 2 min annealing (57.5°C) and 3 min extension (72°C) times were used after a hot start (95°C 5 min, 57.5°C 3 min), followed by 10 min final extension at 72 °C. 5 ⁇ l ofthe first PCR product was used forthe second PCR with forward primers SEQ ID NO.4 for adenovirus and SEQ ID NO. 5 for baculovirus.
  • the reverse primer for both viruses was SEQ ID NO. 6.
  • the same protocol was used as in the Example of WO-A-001/09390, but with 19 cycles. Bands were visualized on 1% agarose gel using ethidium bromide staining. Clinical chemistry analyses
  • lacZ marker gene was mostly found in endothelial cells of the microvessels and in distinct choroid plexus cells in the third ventricle (2 mm from the injection site). Some transgene expression was also seen in the subarachnoidal space. The transgene expression was not detected in other cell types in brain. Clear differences were seen with the adenovirus vector; adenoviruses transduced ventricular ependymal lining and glial cells in corpus callosum with high efficacy. Cells in the subarachnoidal space were also occasionally transduced. Gene expression Transduction efficiency of baculoviruses was as high as 76.8% ⁇ 14 in choroid plexus epithelial cells.
  • transgene expression was highest in the fifth day after the baculovirus transduction.
  • the transgene expression decreased rapidly in two weeks: after 10 days 30.1 % + 6 of the choroid plexus cells were lacZ-positive and after 14 days, only a few positive cells could be detected.
  • a similar time course was seen with adenoviruses: 5 days after the gene delivery, corpus callosum glial cells and ependymal cells were strongly lacZ-positive.
  • the transgene expression decreased in 2-3 weeks, but remained detectable at three weeks timepoint (30.6% ⁇ 13 for corpus callosum and 38.1% ⁇ 12 for ependyma).
  • baculoviruses did not induce a marked microglia response, since only 2 of 16 /acZ-positive rats showed positive immunostaining in the brain.
  • adenovirus delivery elicited a marked microglia response within the transduced tissue.
  • Microglia response was seen in all but one of the analyzed rats. The response increased from moderate to strong from day 5 to day 14 and stayed strong for 21 days. Biodistribution and clinical chemistry
  • transgene expression was found in the fore and hindbrain after the local delivery and in the spleen, heart and lung from rats tested 5 days after the baculovirus gene transfer. After adenovirus injections, transgene expression was seen in forebrain and hindbrain and, from one animal of all tested, in the liver. No major safety problems were found in clinical chemistry analyses, which showed no significant effect of baculoviruses or adenoviruses on acetylaminotranserase, alanineaminotransferase, C-reactive protein, Creatinin, biliribin or Hgb values.

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  • Life Sciences & Earth Sciences (AREA)
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  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP02730459A 2001-05-29 2002-05-28 Gentransfer mittels eines baculoviralen vektors Withdrawn EP1392837A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
WOPCT/GB01/02383 2001-05-29
PCT/GB2001/002383 WO2001090390A1 (en) 2000-05-26 2001-05-29 Use of baculovirus vectors in gene therapy
GB0128620 2001-11-29
GBGB0128620.2A GB0128620D0 (en) 2001-11-29 2001-11-29 Gene delivery of a viral vector
PCT/GB2002/002504 WO2002096469A2 (en) 2001-05-29 2002-05-28 Gene delivery via a baculovirus vector

Publications (1)

Publication Number Publication Date
EP1392837A2 true EP1392837A2 (de) 2004-03-03

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EP02730459A Withdrawn EP1392837A2 (de) 2001-05-29 2002-05-28 Gentransfer mittels eines baculoviralen vektors

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EP (1) EP1392837A2 (de)
WO (1) WO2002096469A2 (de)

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
EP1648948B1 (de) 2003-07-18 2012-02-08 Agency for Science, Technology and Research Wärmeempfindliche polymere für die therapeutische verwendung und herstellungsverfahren

Family Cites Families (4)

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Publication number Priority date Publication date Assignee Title
KR100695590B1 (ko) * 1996-11-01 2007-03-14 아크 테라퓨틱스 리미티드 산화질소 또는 프로스타시클린 생산을 자극하는 약제의 치료학적 용도 및 전달 기구
DE19735593C2 (de) * 1997-08-15 1999-08-26 Hepavec Ag Fuer Gentherapie Hüllprotein-modifizierter Baculovirus-Vektor für die Gentherapie
WO2000005394A1 (fr) * 1998-07-24 2000-02-03 Aventis Pharma S.A. Vecteurs derives de baculovirus et utilisation pour le transfert d'acides nucleiques dans les cellules nerveuses des vertebres
GB0012997D0 (en) * 2000-05-26 2000-07-19 Eurogene Limited Gene delivery

Non-Patent Citations (1)

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Title
See references of WO02096469A3 *

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WO2002096469A2 (en) 2002-12-05
WO2002096469A3 (en) 2003-03-06

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