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WO1998041618A9 - Procede de purification de virus - Google Patents

Procede de purification de virus

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Publication number
WO1998041618A9
WO1998041618A9 PCT/US1998/003879 US9803879W WO9841618A9 WO 1998041618 A9 WO1998041618 A9 WO 1998041618A9 US 9803879 W US9803879 W US 9803879W WO 9841618 A9 WO9841618 A9 WO 9841618A9
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WIPO (PCT)
Prior art keywords
protein
fiber
sequence
vector
capsid
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PCT/US1998/003879
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English (en)
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WO1998041618A1 (fr
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Priority to AU64429/98A priority Critical patent/AU6442998A/en
Publication of WO1998041618A1 publication Critical patent/WO1998041618A1/fr
Publication of WO1998041618A9 publication Critical patent/WO1998041618A9/fr

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  • the instant invention relates generally to vectors and methods for gene therapy, and more particularly, to an adenovirus vector containing a heterologous peptide epitope in the HI loop of the vector fiber knob and methods for purifying such a vector for application in transgene delivery.
  • Viruses particularly adenoviruses have significant use as vectors for carrying genetic material into cells.
  • adenoviral vectors are widely used in experimental systems, and it is anticipated that they will soon have a significant use in a number of genetic therapies, both in humans and animals.
  • the viral vectors In order to increase the efficiency of such viral-based genetic therapies, and to minimize the side effects resultant therefrom, the viral vectors must be concentrated and/or purified.
  • Recombinant adenovirus vectors have found wide employment for a number of gene therapy applications (21, 35, 38). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo. Indeed, recombinant adenovirus vectors are distinguished from other available systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 20, 25, 27, 29, 31). Despite this property, specific aspects of the adenovirus biology have prevented the full realization of the potential of such vectors. In this regard, the broad tropism profile of the parent virus for cells of diverse tissues potentially allows unrestricted gene delivery.
  • the promiscuous tropism of the adenovirus vector represents a confounding factor.
  • strategies to modify the native tropism of adenovirus have been developed to allow the derivation of vectors capable of targeted gene delivery.
  • Adenoviruses of serotypes 2 and 5 normally achieve initial recognition and binding to target cells by means of interactions between the carboxy-terminal knob domain of the fiber protein and the primary receptor (4, 17, 36). After binding, RGD motifs in the penton base interact with cellular integrins of the ⁇ v ⁇ 3 and v ⁇ 5 types (1-3, 37, 39, 40). This interaction triggers cellular internalization whereby the virions achieve localization within the endosome. Acidification of the endosome elicits conformational changes in capsid proteins, allowing their interaction with the endosome membrane in a manner that achieves vesicle disruption and particle escape. Following endosomolysis, the virion translocates to the nucleus, where the subsequent steps of the viral life cycle occur. This understanding of the key role played by capsid proteins in the viral infectious pathway has suggested strategies to alter this process via modifications of these proteins.
  • the HI loop possesses a number of features which predict its utility as an alternative site for ligand incorporation. Specifically, the HI loop does not contribute to intramolecular interactions in the knob, therefore incorporation of additional protein sequence should not affect the trimerization of the fiber. In addition, the loop consists mostly of hydrophilic amino acid residues and is exposed outside the knob. It thus potentially demonstrates a high degree of flexibility, creating an optimal environment for ligand incorporation. Furthermore, the lengths of HI loops vary significantly in knobs of different adenovirus serotypes. This fact suggests that alterations of the original structure of the loop, such as insertions and deletions, should be compatible with the correct folding of the entire knob domain. Finally, the HI loop is not involved in the formation of the putative cell-binding site localized in the knob.
  • the instant invention develops a novel approach to modifying the adenovirus fiber protein by employing the HI loop of the knob for this purpose.
  • This invention shows that it is possible to incorporate heterologous amino acid sequences into the HI loop without affecting the correct folding of the fiber polypeptide and its biological functions. Further, this locale offers advantages for strategies designed to achieve tropism modification based upon genetic alteration of capsid proteins.
  • Fig. 1 shows modifications of the HI loop of the fiber knob. PCR-based mutagenesis was employed to delete a portion of the fiber gene encoding the hypervariable region of the HI loop. A unique EcoR V restriction site was incorporated in place of the deletion to allow the cloning of segments of DNA coding for heterologous protein sequences. In the fiber-FLAG protein, deleted amino acids of the HI loop were restored, and FLAG octapeptide was incorporated between threonine-546 and proline-547. The site of deletion is indicated by a filled triangle.
  • Fig. 2 shows analysis of recombinant fiber proteins by polyacrylamide gel electrophoresis.
  • Fiber proteins expressed in insect cells were analyzed by gel electrophoresis to confirm their trimeric configurations. To dissociate trimers to monomers, the proteins were denatured by boiling them in the sample buffer prior to loading them on a 7.5% polyacrylamide gel. The bands were visualized by Coomassie blue staining.
  • A Six-histidine-tagged fiber proteins purified on an Ni-NTA-Sepharose column. Lane 1, wild type fiber, boiled; lane 2, wild type fiber, unboiled; lane 3, fiber-FLAG, boiled; lane 4, fiber-FLAG, unboiled; lane M, broad-range protein standards.
  • B Fiber-FLAG protein purified by immunoprecipitation with anti-FLAG M2 affinity gel. Lane 1, unboiled protein; lane 2, boiled protein; lane M, broad-range protein standards. The numbers on the left indicate molecular masses of marker proteins in kilodaltons.
  • Fig. 3 shows inhibition of adenovirus infectivity by recombinant fiber proteins.
  • HeLa cells were preincubated with either the wild-type (wt) fiber (A) or fiber-FLAG (B) at the indicated concentrations for 10 min at room temperature.
  • AdCMVLuc was then added at a multiplicity of infection of 10, and incubation was continued for another 30 min at room temperature.
  • the unbound virus was aspirated, complete medium was added, and the cells were transferred to 37 °C. After 30 h the cells were lysed and luciferase activity was determined. Luciferase activities are given as percentages of the activity in the absence of blocking fiber protein. Each point represents the mean of four determinations obtained in one experiment.
  • Fig. 4 shows generation of Ad5F ffl FLAG.
  • the master plasmid, pTG3602 was modified to incorporate a unique Swal restriction site in the fiber gene, thereby creating plasmid pVK50, suitable for fiber modifications.
  • the genome of Ad5F HI FLAG was generated by homologous DNA recombination in E. coli between the DNA fragment containing the fiber-FLAG gene and plasmid pVK50 linearized by Swal digestion.
  • the resulting plasmid, pVK300 which contains the complete adenoviral genome with a modified fiber gene, was cleaved with Pad and was then used to transfect 293 cells.
  • Fig. 5 shows adenovirus binding assay results.
  • Aliquots of A549 cells containing 10 5 cells per sample were incubated for 1 h at 4°C with serial dilutions of either wild-type (wt) Ad5 fiber or fiber-FLAG (see Materials and Methods).
  • Virions of Ad5CMVLacZ (A) and Ad5F m FLAG (B) labeled with 125 I were added to samples, and incubation was continued for an additional hour.
  • the cells were washed with 4 ml of PBS containing 0.1% BSA and pelleted by low-speed centrifugation. Radioactivities of samples were determined with a gamma counter. Each point represents the mean of two determinations obtained in one experiment.
  • Fig. 6 shows accessibility of the FLAG peptide in the context of intact Ad5F HI FLAG virions.
  • Virions of Ad5F ffl FLAG purified on a CsCl gradient were dialyzed, immunoprecipitated with anti-FLAG M2-affinity gel as described herein, and eluted from the gel with free FLAG peptide.
  • Recombinant adenovirus vector Ad5CMVLuc containing unmodified fiber was used as a negative control for binding. Aliquots of all the fractions collected throughout the purification procedure were treated with DNase I to digest traces of the cellular DNA and then treated with SDS, EDTA, and proteinase K to release adenovirus DNA from the virions.
  • Lanes 1 through 3 AdCMVLuc in the supernatant containing unbound material, buffer wash, andFLAG-eluate, respectively; lanes 4 through 6, Ad5F ffl FLAG in the supernatant, buffer wash, and FLAG-eluate, respectively.
  • Lane M DNA molecular weight standards (the bands corresponding to marker fragments ranging from 3 to 12 kb are seen on the
  • the capsid of a gene therapy vector plays a significant role in targeting the type of host cell a vector will transfect.
  • Modification regions exposed to the vector environment serves to direct a vector towards receptors specific to a well defined host cell population. Furthermore, such a modification also serves as a molecular recognition site for purification of modified vectors from a mixed population of vectors.
  • the invention is described in reference to adenovirus capsid protein modification, to incorporate a heterologous epitope into the protein in order to ablate the inherent tropism of the virus, it is appreciated that the methods, protocols and reagents herein are broadly applicable to protein coated gene therapy vectors.
  • Protein coated gene therapy vectors are defined herein to include viruses, virions, amd mono- and bi-layer micelles having proteins attached thereto.
  • the HI loop of the fiber knob as an alternative site for incorporation of heterologous peptide sequences.
  • the HI loop does not contribute to interactions within the knob which stabilize its trimeric configuration and is not involved in the formation of the receptor binding site.
  • the HI loop is exposed outside the knob, thereby facilitating the interaction of potential ligand with the cellular receptor.
  • a FLAG coding sequence was incorporated into the region of the fiber gene corresponding to the HI loop and expressed this modified gene in baculovirus infected insect cells.
  • An amino terminal six-His tag incorporated into the design was used for simple chromatographic purification of recombinant fiber protein. Baculovirus-directed expression of this recombinant full size fiber was efficient, and according to our gel analysis and ELISA with the trimer-specific anti-fiber monoclonal antibody MAb, the product of expression was trimeric.
  • a recombinant adenovirus genome was generated by using a novel method described recently (7).
  • a master plasmid, pTG3602 from Transgene was modified to engineer a vector which greatly facilitates modifications of the fiber gene in the adenovirus genome.
  • Ad5F ffl FLAG the virus of interest
  • the HI loop of the fiber knob is a convenient site for incorporation of heterologous peptide ligands which may be successfully utilized in order to target adenovirus vectors for gene therapy applications.
  • This location in the knob can be used either as an alternative site or in addition to carboxy-terminal modifications of the fiber protein, offering a unique loop-like environment, which may be required for proper biological functioning of some ligand sequences.
  • this structure may be beneficial for peptide ligands obtained from phage display libraries containing random peptide sequences flanked with two cysteine residues forming a disulfide bridge (23, 24).
  • ligands with the loop-like configuration may be less susceptible to degradation by cellular carboxypeptidases than ligands positioned at the carboxy terminus of the fiber.
  • recombinant adenoviruses containing different targeting moieties in this locale are created.
  • Generation of recombinant adenoviruses containing fibers with targeting ligands incorporated into the HI loop of the knob will facilitate further efforts towards an improved adenovirus vector for gene therapy applications.
  • the successful use of the FLAG epitope in binding experiments suggests that this or a similar purification tag can be incorporated into an adenovirus virion to facilitate its purification. This simple purification technique occurs at atmospheric laboratory pressure and does not require expensive laboratory equipment such as ultracentrifuges or high pressure liquid chromatography systems and can be easily scaled up if needed.
  • the present invention provides a method for purifying and concentrating virus, and the method is particularly suited for use with adenoviral vectors.
  • an adenoviral vector is modified so that its protein capsid expresses one or more heterologous epitopes thereupon. These foreign epitopes distinguish the modified adenovirus from the corresponding, unmodified virus and provide an active site which is capable of binding to an antibody, chelating agent, other chemical reagent or the like.
  • the modified virus is contacted with an appropriate binding reagent, which itself is preferably immobilized, as for example in a chromatography column. The modified virus is then bound and retained, and may be collected by freeing from the agent.
  • an appropriate binding reagent may be immobilized on a support medium in a column, and process liquid including the virus may then be flushed through the column.
  • the column will selectively bind the modified virus, while permitting the unmodified virus, and other materials to pass therethrough.
  • the modified virus may then be eluted from the column by flushing with an appropriate reagent. In this manner, a purified, concentrated sample of the adenoviral vector is obtained.
  • the protein capsid of the virus is preferably modified by utilizing a plasmid to insert genetic information into the adenovirus to cause it to express the heterologous epitope thereupon.
  • modification of the capsid will be at a carboxy terminal of the capsid fibers.
  • the modification will take place at the knob domain of the fiber; most preferably at the H-I loop of the knob domain.
  • a plasmid vector designated pTG3602S is utilized to generate recombinant adenoviral genomes which contain genes encoding: fiber-short linker-RGS6H; fiber-short linker-somatostatin; in the capsids of adenovirus.
  • This sequence was expressed at the C-terminus of the capsid fiber of an adenovirus designated Ad5FcRGS6H.
  • Ad5FcRGS6H adenovirus designated Ad5FcRGS6H.
  • the presence of the modified fiber gene in the viral genome was confirmed by a cycle sequencing readout on viral DNA isolated from purified virions.
  • the presence of C-terminal modifications in the fibers was confirmed by Western blot analysis carried out on purified virus.
  • AdCMV-Luc did not adhere to the column and was washed therefrom by the 40mM imidazole, but that the Ad5FcRGS6H modified virus bound to the column and was only eluted therefrom by the 0.5M imidazole.
  • plasmids may be employed to modify the capsid of a virus to produce a heterologous epitope thereupon, and that this modified virus may be separated from other virus by use of an affinity reagent.
  • the adenovirus vector may be made to selectively bind to an appropriate affinity reagent, by modifying the protein capsid thereof so as to cause the capsid to have a novel epitope thereupon.
  • the virus may be selectively separated from unmodified virus, and other materials.
  • the methodology of the present invention may be employed to collect, concentrate and purify viral materials through a process which is simple, low cost and reliable. Furthermore, the process is compatible with standard techniques used to prepare adenoviral vectors. Consequently, the present invention greatly facilitates the production of materials for a variety of genetic therapies.
  • Example 1 Cells.
  • T4 DNA ligase T4 polynucleotide kinase
  • proteinase K was from either New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.).
  • the concentrations of purified proteins were determined by the Bradford protein assay (Bio-Rad, Hercules, Calif.) with bovine gamma globulin as the standard.
  • Anti-fiber monoclonal antibodies 4D2 (19) and 1D6.14 (14) were generated at the University of Alabama at Birmingham Hybridoma Core Facility.
  • Anti-FLAG monoclonal antibody M2 and M2-affmity gel were purchased from Scientific Imaging Systems (Eastman Kodak, New Haven, Conn.)
  • Example 5 Construction of recombinant plasmids.
  • a PCR technique was employed. Two pairs of primers FI (5' TAAGGATCCG GTGCCATTAC AGTAGGAAAC AAAAATAA 3') and Rl (5' CATAGAGTAT GCAGATATCG TTAGTGTTAC AGGTTTAGTT TT G 3'), and F2 (5' GTAACACTAA CGATATCTGC ATACTCTATG TCATTTTCAT GG 3') and R2 (5' CCCAAGCTTA CAATTG AAAA ATAAACACGT TGAAACATAA C 3'), were used to amplify portions of the fiber gene corresponding to positions 1159 to 1451 and 1642 to 1747, respectively.
  • the second fragment also contains 43 bp of Ad5 DNA adjacent to the 3' end of the fiber gene in the viral genome.
  • the fragments generated were then gel purified, mixed, and joined by the third PCR using primers FI and R2.
  • the product obtained contains a unique EcoRV restriction site in place of the deleted portion of the sequence encoding the HI loop, as well as BamHI and Hindlll sites inserted into the ends of the molecule to facilitate subsequent cloning.
  • This DNA fragment was cleaved with BamHI and Hindlll and cloned into the BamHI-Hindlll-digested bacterial expression vector pQE30 (Qiagen, Santa Clara, Calif), resulting in plasmid pQE.KNOB ⁇ HI.
  • oligonucleotides TACACTAAAC GGTACCCAGG AAACAGG AGA CACAACTGACT ACAAGGA CGACGATGACAAG CC and GGCTTGTCATC GTCGTCCTTG TA GTCAGTTGT GTCTCCTG TTT CCTGGGTACCG TTTAGTGTA were annealed to form a duplex and cloned into EcoR F-digested pQE.KNOB ⁇ HI.
  • the plasmid containing the duplex in the correct orientation was designated pQE.KNOB m FLAG.
  • the transfer plasmids for the generation of recombinant baculoviruses expressing chimeric fibers were made as follows: a Bglll-Mfel fragment from pQE.KNOB m FLAG was utilized to replace the Bglll-Mfel fragment in the vector pBS.F5.UTR which has been described previously (24), thereby generating pBS.F5 M FLAG. A BssHII-Xhol fragment from pBS.FSmFLAG was then cloned into the BssHII-XhoI-digested baculovirus transfer vector pFastBacl (Life Technologies, Gaithersburg, Md.), resulting in pFB.F5 H ,FLAG.
  • the BamHI-BssHII fragment of pFB.F5 HI FLAG was replaced with a synthetic duplex made with oligonucleotides GATCCATGCA TCACCATCAC C ATCACAAG and CGCGCTTGTG ATGGTGATGG TGATGCATG, which encodes MetHis 6 Lys.
  • the resultant plasmid, pFB6H.F5 HI FLAG contains the gene coding for a fiber with an amino-terminal six-His tag and FLAG peptide inserted into the HI loop.
  • Plasmid pTG3602 was partially digested with Ndel and ligated with an Ndel-Swal linker, TACCCATTTAAATGGG. This plasmid, containing a Swal site in the fiber gene was designated pVK50.
  • a recombinant adenovirus genome containing a gene encoding the fiber-FLAG protein was generated by homologous DNA recombination in E. coli BJ5183 between pVK50 linearized with Swal and the 3-kb ⁇ coRI fragment from pN ⁇ B.F5 HI FLAG containing the gene of interest, as described by Chartier et al. (7).
  • the newly generated genome was then excised from the resultant plasmid, pVK300, and employed to rescue the virus.
  • Ad5 vectors AdCMVLuc and AdCMVLacZ, which express firefly luciferase and bacterial ⁇ -galactosidase (18), respectively, were obtained from R. D. Gerard, the University of Texas Southwestern Medical Center, Dallas, Tex.
  • Ad5F HI FLAG was generated by transfection of 293 cells with ⁇ ci-digested pVK300, as previously described (7). Adenoviruses were propagated on 293 cells and purified by centrifugation in CsCl gradients according to a standard protocol (15). Determination of virus particle titer was accomplished spectrophotometrically by the method described by Maizel et al. (28), with a conversion factor of 1.1 x 10 12 viral particles per absorbance unit at 260 nm. To determine the titer of infectious viral particles on 293 cells, a plaque assay was employed as described by Mittereder et al. (32).
  • Recombinant baculoviruses expressing chimeric fibers were generated with a Bac-to-Bac expression kit from Gibco-BRL (Life Technologies) according to the manufacturer's protocol.
  • Example 7 Expression and purification of six-His-tagged recombinant proteins.
  • Recombinant fibers were expressed in Spodoptera frugiperda Sf9 cells infected with recombinant baculovirus by the method recommended for the Bac-to-Bac system
  • NTA Ni-nitrilotriacetic acid
  • Qiagen Ni-nitrilotriacetic acid
  • an enzyme-linked immunosorbent assay (ELISA) was employed.
  • the six-His tagged fibers were immobilized on Ni-NTA HisSorb Strips (Qiagen) essentially as described in the Qiagen manual. 200 ⁇ of fiber protein solution at a concentration 1 ⁇ g/ml was added to each well of an Ni-NTA HisSorb Strip and incubated for 1 h at room temperature. After incubation the wells were washed four times with phosphate-buffered saline (PBS)-Tween buffer, and 200 ⁇ l of anti-fiber antibody (1 :2000 dilution) or anti-FLAG antibody (1 : 140 dilution) was added.
  • PBS phosphate-buffered saline
  • Ad5F m FLAG or AdCMVLuc purified on CsCl gradients were dialyzed against HEPES buffer (10 mM HEPES, 1 mM MgCl 2 , 10% glycerol, [pH 7.4]) and absorbed onto M2-affinity gel (Eastman Kodak) as follows.
  • the supernatant containing unbound material, the wash, and the eluate were then employed to detect the presence of the virus. For this, aliquots of these fractions were treated for 1 h at 37 °C with sodium dodecyl sulfate, EDTA, and proteinase K at final concentrations of 1%, 10 mM and 100 ⁇ g/ml, respectively. The samples were analyzed by agarose gel electrophoresis to detect viral DNA.
  • Example 10 Purification of the fiber-FLAG protein by immunoprecipitation.
  • the recombinant fiber-FLAG protein was expressed in baculovirus infected Sf9 cells as follows. For large scale expression of the fiber-FLAG protein, monolayers of Sf9 cells in T75 flasks were infected with recombinant baculovirus at multiplicity of infection of 5 to 10 and then were incubated at 28 °C until a complete cytopathic effect was observed.
  • the cells were scraped, pelleted by low speed centrifugation, and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.02% sodium azide, 100 ⁇ g/ml phenylmethylsulfonyl fluoride, 1 ⁇ g of aprotinin per ml).
  • lysis buffer 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.02% sodium azide, 100 ⁇ g/ml phenylmethylsulfonyl fluoride, 1 ⁇ g of aprotinin per ml.
  • the cells were then incubated on ice for 30 min.
  • the lysate was cleared by centrifugation at 12,000 x g for 5 minutes in a microcentrifuge.
  • the cleared lysate was mixed with the
  • Example 11 Trimerization assay of recombinant proteins.
  • these proteins were analyzed by SDS-polyacrylamide gel electrophoresis as previously described (30). Proteins were either boiled prior to electrophoresis, to dissociate the trimers, or loaded on the gel without denaturation. The trimeric or monomeric configuration of these molecules was thus determined based on their mobilities in the gel.
  • Example 12 Inhibition of virus mediated gene transfer by recombinant fiber proteins.
  • Example 13 Virus binding assay.
  • the recombinant adenoviruses AdCMVlacZ and Ad5F HI FLAG were purified on a CsCl gradient and dialyzed against buffer containing 10 mM HEPES, 1 mM MgC12, 10%) glycerol [pH 7.4]. Aliquots of both viruses containing 50 ⁇ g of viral protein were labeled with 125 I using IODO-BEADS iodination reagent (Pierce, Rockford, 111.) as previously described (20). Labeled viruses were purified from unincorporated 125 I by gel filtration on PD-10 columns (Pharmacia, Piscataway, N.J.). Fifty-microliter aliquots of labeled virions with total radioactivities of 10 5 cpm were then added to A549 cells preincubated with fiber dilutions or PBS and incubated at 4°C for another hour.
  • Example 14 Expression of recombinant fiber knobs in E. coli.
  • the deletion made removed the portion of the Hl-loop which varies most significantly in the fiber knobs of different serotypes of human adenoviruses.
  • the sequence generated by PCR contained an open reading frame corresponding to two segments of the fiber protein including amino acids glycine- 387 through isoleucine-534 and serine-548 through glutamine-581 (here and in the following text the coordinates are given according to the wild type Ad5 fiber protein sequence). This sequence was cloned into bacterial expression vector, pQE30, thereby creating a fusion gene encoding the deleted fiber knob and containing amino terminal 6His purification tag.
  • Example 15 Characterization of recombinant fibers expressed in baculovirus infected insect cells.
  • a PCR approach was used to derive a gene encoding the Ad5 fiber knob with a partial deletion in the HI loop.
  • This deletion was engineered to remove amino acids TLNGTQETGDTTP from the HI loop of the fiber knob domain and to introduce a unique EcoRV site in place of the deleted sequence, thereby facilitating the cloning of alternative sequences in this region (Fig. 1).
  • the deletion removed the portion of the HI loop which varies most significantly in the fiber knobs of different serotypes of human adenoviruses.
  • the sequence generated by PCR contained an open reading frame corresponding to two segments of the fiber protein including amino acids glycine-387 through isoleucine-534 and serine-548 through glutamine-581 (coordinates given are according to those of the wild type Ad5 fiber protein sequence).
  • This sequence was cloned into the plasmid vector pQE30.
  • the newly generated plasmid, pQE.KNOB ⁇ HI was then utilized as a cloning vector to incorporate a fragment of DNA encoding the FLAG octapeptide (DYKDDDDK), which has been widely used as a detection and purification tag in a variety of studies.
  • this FLAG peptide was chosen in fiber constructs as a probe to determine whether a heterologous peptide sequence incorporated into the HI loop of the knob was accessible in the context of a trimeric fiber molecule.
  • the FLAG coding sequence was introduced as insertions between threonine-546 and proline-547. This plasmid was then employed to construct a full-size recombinant fiber gene in a baculovirus transfer vector.
  • a similar transfer plasmid containing the wild type fiber gene was designed for control purposes.
  • Recombinant fibers were recovered from the lysates of baculovirus-infected insect cells with Ni-NTA-Sepharose designed for purification of the six-His-tagged proteins.
  • the yield of purified fibers was in the range of 10 ⁇ g of protein per 10 6 infected cells.
  • Analysis by SDS-polyacrylamide gel electrophoresis of both recombinant proteins showed that they formed stable trimers which, when boiled in the gel loading buffer, dissociated into monomers of the expected molecular mass of 63 kDa (Fig. 2 A). This result demonstrated that the incorporation of a short peptide sequence in the HI loop of the knob does not ablate trimerization of the fiber. Therefore, by using the baculovirus expression system we were able to obtain preparative amounts of the recombinant fibers of interest which were suitable for subsequent assays.
  • Example 16 Accessibility of the FLAG peptide in the context of trimeric fiber.
  • the fiber-FLAG protein efficiently bound to M2-affinity gel, demonstrating the availability of the FLAG epitope for interaction with an anti-FLAG monoclonal antibody in the context of the trimeric fiber molecule.
  • this interaction did not affect the stability of the trimer, suggesting that a recombinant virion containing a novel ligand incorporated in the HI loop of the fiber knob will maintain its structural integrity throughout the binding step of the infection.
  • Example 17 Inhibition of adenovirus infection by recombinant fiber-FLAG protein.
  • a fiber-FLAG recombinant protein was employed to block adenovirus infection in the in vitro setting. This established assay is based on the fact that recombinant adenovirus fiber proteins are capable of blocking infection by the adenovirus from which they were derived. In addition, this inhibition of viral infection takes place in a dose dependent manner.
  • HeLa cells seeded in 12-well tissue culture plates were preincubated with various concentrations of the wild-type Ad5 fiber or fiber-FLAG protein prior to infection with the recombinant Ad5 vector AdCMVLuc, which expresses firefly luciferase as a reporter.
  • Example 18 Characterization of the fiber-FLAG protein by ELISA.
  • this recombinant protein by ELISA was analyzed, employing several monoclonal antibodies specific for the FLAG epitope and different conformations of the Ad5 fiber.
  • wild type fiber and fiber-FLAG proteins expressed in insect cells were absorbed on HisSorb ELISA strips covered with Ni-NTA (Qiagen) and probed with anti-fiber antibody 4D2 or 1D6.14 or anti-FLAG antibody M2.
  • Antibody 4D2 reacts with Ad5 fiber monomers and trimers and was used in this assay as a positive control, whereas antibody 1D6.14 binds to an as yet unidentified conformational epitope in the fiber knob and is trimer specific.
  • the ELISA strips were then developed with goat anti-mouse antibody-HRP conjugate.
  • this method involves recombination between two linear DNA molecules cotransformed into bacterial cells to generate a recombinant adenovirus genome.
  • One of these molecules is plasmid pTG3602, or its derivative, containing the full-size adenovirus genome cloned in the bacterial vector and flanked with two Pad sites.
  • the second partner in this recombination scheme is the genetic construct of interest flanked with two segments of adenovirus genomic DNA which dictate the localization of this construct in the adenovirus genome generated as a result of the recombination.
  • This DNA sequence can be either a transgene or the original Ad5 gene, modified by traditional methods of genetic engineering in the context of small recombinant plasmids.
  • this plasmid was cleaved with a restriction enzyme within or near the region of the genome where the final construct was going to be inserted.
  • this method has numerous advantages compared to traditional generation of recombinant adenovirus genomes by homologous recombination in mammalian cells, it requires the existence of unique restriction sites within the regions of the adenovirus genome to be modified.
  • Ad5 genomic DNA in pTG3602 does not contain any unique restriction sites in the fiber gene, which limits its utility for modifications of fiber.
  • this plasmid was modified by inserting a unique cleavage site for the restriction endonuclease Swal into the fiber gene.
  • a unique cleavage site for the restriction endonuclease Swal was converted into Swal site by insertion of an Sw ⁇ i-linker (Fig. 4).
  • the plasmid generated, pVK50 was then utilized for homologous recombination with the fragment of DNA containing the gene encoding fiber-FLAG flanked with viral DNA adjacent to the fiber gene in the Ad5 genome.
  • pVK300 a plasmid, pVK300, containing a modified fiber gene in the context of the complete adenovirus genome was derived.
  • Adenovirus DNA was released from pVK300 by Pad digestion and used for transfection of 293 cells to rescue the virus as described previously (7).
  • Ad5F ffl FLAG DNA isolated from CsCl gradient-purified virions of the newly generated virus, Ad5F ffl FLAG, was subjected to PCR analysis and cycle sequencing to confirm the presence of the FLAG coding sequence in the fiber gene inco ⁇ orated in the genome. According to both analyses, Ad5F m FLAG indeed contained the fiber gene of interest.
  • Example 20 Characterization of Ad5F HI FLAG by a cell-binding assay.
  • Example 22 Characterization of Ad5F HI FLAG by antibody binding. Since the ultimate goal of our strategy is engineering of a targeting ligand into the knob, it was necessary to determine whether such a ligand would be available for interaction with its target cell surface receptor after inco ⁇ oration into the adenoviral virion. To this end, the FLAG sequence inco ⁇ orated into the fiber of Ad5F ffl FLAG was employed to test the accessibility of the Hl-loop of the knob in a context of intact adenoviral particle. This was accomplished in an assay similar to the one used to evaluate FLAG accessibility in the recombinant fiber-FLAG protein expressed in insect cells.
  • Virions of purified on CsCl gradient were dialyzed against Hapes buffer and incubated with M2 affinity gel to allow interaction between FLAG peptide and anti- FLAG monoclonal antibody conjugated to the gel matrix.
  • prepared virons of AdCMVLuc containing wild type fibers were utilized in this experiment as a negative control. After incubation the buffer containing unbound material was collected and the gel was washed with the buffer to remove the traces of free virus. Finally, the viruses were eluted from the gel by soluble FLAG peptide. Aliquots of the samples collected were treated with proteinase K to release viral DNA from virions which was then visualized by agarose gel electrophoresis (Figure 5).
  • Example 22 FLAG accessibility in the context of the Ad5F HI FLAG virion.
  • the FLAG sequence inco ⁇ orated into the fiber of Ad5F ffl FLAG was employed to test the accessibility of the HI loop of the knob in the context of an intact adenovirus particle. This was accomplished in an assay similar to the one used to evaluate FLAG accessibility in the recombinant fiber-FLAG protein expressed in insect cells.
  • Virions purified on a CsCl gradient were dialyzed against HEPES buffer and incubated with M2-affinity gel to allow interaction between the FLAG peptide and an anti-FLAG monoclonal antibody conjugated to the gel matrix.
  • prepared virions of AdCMVLuc containing wild-type fibers were utilized in this experiment as a negative control. After incubation, the buffer containing unbound material was collected and the gel was washed with the buffer to remove the traces of free virus. Finally, the viruses were eluted from the gel with soluble FLAG peptide. Aliquots of the samples collected were treated with proteinase K to release viral DNA from virions, which was then visualized by agarose gel electrophoresis (Fig. 7).
  • AdCMVLuc did not react with M2 antibody and were detected only in the fraction containing unbound virus and in the wash.
  • Ad5F HI FLAG particles efficiently bound to the M2-affinity gel, since viral DNA was present primarily in the FLAG peptide eluate.

Abstract

Elargissement de l'utilité des vecteurs d'adénovirus recombinants courants dans les applications de thérapie génique par la création de vecteurs ciblés capables de transporter des gènes dans des types sélectionnés de cellules in vivo. Pour produire ce ciblage, l'incorporation de ligands dans la protéine fibreuse adénovirale, dans laquelle la protéine suscite la liaison primaire de l'adénovirus au récepteur de sa surface cellulaire, est réalisée en utilisant la boucle HI de la protubérance fibreuse comme site adéquat pour l'incorporation de ligands hétérologues. Des protéines fibreuses recombinantes exprimées dans diverses cellules, notamment dans des cellules d'insectes infectées par baculovirus et E. coli montrent que l'incorporation de l'octapeptide FLAG dans la boucle HI ne supprime pas la trimérisation de la fibre et ne perturbe pas la formation du site de liaison cellulaire localisé dans la protubérance. Un adénovirus conforme à l'invention présentant cette fibre modifiée démontre qu'une courte séquence peptidique formée dans la protubérance est compatible avec les fonctions biologiques de la fibre. Dans les virions matures contenant des fibres modifiées, un peptide incorporé dans la protubérance conformément à l'invention reste capable d'établir des liaisons. L'invention décrit l'incorporation des ligands hétérologues dans la boucle HI de la protubérance fibreuse, les propriétés de ce site étant compatibles avec son utilisation dans des stratégies de reciblage des adénovirus.
PCT/US1998/003879 1997-03-14 1998-03-13 Procede de purification de virus WO1998041618A1 (fr)

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