| Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease -> Monitor Keywords |
|
|
 
Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus proteaseRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophageEfficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060210965, Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a Divisional of U.S. patent application Ser. No. 09/843,949 filed Apr. 30, 2001, the entire content of which is incorporated by reference in this application. FIELD OF THE INVENTION [0002] The present invention relates to a method of generation of adenovirus recombinant vectors and adenovirus-based expression libraries, in particular to a method of generation of adenovirus recombinant vectors and adenovirus-based expression libraries by positive selection of adenovirus recombinant vectors through ectopic expression of the adenovirus protease. BACKGROUND OF THE INVENTION [0003] The term "gene therapy" is usually understood to mean the process in which a gene is introduced into the somatic cells of an individual with the aim of being expressed in the cells, to produce some therapeutic effect. Initially this principle was applied to cases where an additional normal copy of a defective gene was provided to restore the synthesis of a missing protein, such as an enzyme. The concept of gene therapy has since been broadened to include several other approaches. In particular, the transferred gene (transgene) may code for a protein that is not necessarily missing but that may be of therapeutic benefit and difficult to administer exogenously, for example IL-2 or antitumor cytokines. This form of gene therapy aims to enhance in vivo production of potentially therapeutic proteins. This approach is similar to gene vaccination, where the transferred gene is introduced into the cells to express a protein acting as an antigen inducing a protective immune response of the host's immune system. Another form of gene therapy involves transferring into cells non-physiological sequences which have antiviral activity, such as antisense oligonucleotides or sequences. Finally, so-called suicide genes can be transferred into undesirable cells (cancer cells or infected cells), to sensitize them to specific substances. When these substances are administered subsequently, they trigger selective destruction of the targeted cells. [0004] Gene delivery systems which transfer the desired gene into the target cells are based either on physico-chemical or on biological methods. In each case the desired gene can be transferred into cells either in vitro, by extracting cells from an organ and reintroducing the cells transfected in vitro into the same organ or organism, or in vivo, i.e., directly into an appropriate tissue. Known physico-chemical methods of transfection include, for example, gene gun (biolistics), in situ naked DNA injections, complexes of DNA with DEAE-dextran or with nucleic proteins, liposomal DNA preparations, etc. Biological methods, considered to be a more reliable alternative to physico-chemical methods, rely on infectious agents as gene transfer vectors. In this group of methods, viruses have become infectious agents of choice, due to their inherent capability of infecting various cells. The transfer of a foreign gene by a viral vector is known as transduction of the gene. [0005] Several virus classes have been tested for use as gene transfer vectors, including retroviruses (RSV, HMS, MMS, etc.), herpesviruses (e.g., HSV), poxviruses (vaccinia virus), adenoviruses (Ad, mainly derived from type 5 and 2 Ad) and adeno-associated viruses (MV). Of those, adenovirus-based vectors are presently considered to be among the most promising viral vectors, due to their following properties, some of which are unique to this group of vectors: (i) adenovirus vectors do not require cell proliferation for expression of adenovirus proteins (i.e., are effective even in cells at the resting phase); (ii) adenovirus vectors do not integrate their DNA into the chromosomes of the cell, so their effect is impermanent and is unlikely to interfere with the cell's normal functions; (iii) adenovirus vectors can infect non-dividing or terminally differentiated cells, so they are applicable over a wide range of host cells; (iv) adenovirus vectors show a transducing efficiency of almost 100% in a variety of animal cultured cells and in several organs of various species in vivo; (v) adenovirus vectors usually possess an ability to replicate to high titer, a feature important for the preparation of vector stocks suitable for the achievement of efficient transduction in vivo; (vi) adenovirus vectors can accommodate large inserts of exogenous DNA (have a high cloning capacity); (vii) recombination events are rare for adenovirus vectors; (viii) there are no known associations of human malignancies or other serious health problems with adenovirus infections; (adenovirus type 5 is originally known to cause cold conditions in humans; live adenovirus of that type having the ability to replicate has been safely used as a human vaccine (Top et al., J.I.D., 124, 148-154; J.I.D., 124, 155-160(1971)). [0006] Structurally, adenoviruses are non-enveloped viruses, consisting of an external capsid and an internal core. Over 40 adenovirus subtypes have been isolated from humans and over 50 additional subtypes from other mammals and birds. All adenoviruses are morphologically and structurally similar, even though they differ in some properties. Subtypes of human adenoviruses are designated according to serological response to infection. Of those, serotypes Ad2 and Ad5 have been studied most intensively, and used for gene transfer purposes since the 80s. Genetically, adenovirus is a double-stranded DNA virus with a linear genome of about 36 kb. The genome is classified into early (E1-E4) and late (L1-L5) transcriptional regions (units). This classification is based on two temporal classes of viral proteins expressed during the early (E) and late (L) phases of virus replication, with viral DNA replication separating the two phases. [0007] A viral gene transfer vector is a recombinant virus, usually a virus having a part of its genome deleted and replaced with an expression cassette to be transferred into the host cell. Additionally to a foreign (exogenous) gene, the expression cassette comprises components necessary for a proper expression of the foreign gene. It contains at least a promoter sequence and a polyadenylation signal before and after the gene to be expressed. Other sequences necessary to regulate or enhance the gene expression can be included in the cassette for specific applications. [0008] The deletion of some parts of the viral genome may render the virus replication-incompetent, i.e., unable to multiply in the infected host cells. This highly desirable safety feature of viral vectors prevents the spread of the vector containing the recombinant material to the environment and protects the patient from an unintended viral infection and its pathological consequences. The replication-defective virus requires for its propagation either a complementing cell line (packaging cell line) or the presence of a helper virus, either of which serves to replace (restore in trans) the functions of the deleted part or parts of the viral genome. As it has been shown that the production of recombinant viral vectors free of replication-competent helper virus is difficult to achieve, the use of packaging cell lines for the propagation of replication-incompetent viral vectors is considered to be the best choice for gene therapy purposes. [0009] Early adenovirus vectors (sometimes referred to as first generation adenovirus vectors, or singly deficient vectors) relied on deletions (and insertions) in coding regions E1 and/or E3 of the viral genome (see, for example, U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,698,202; U.S. Pat. No. 5,731,172). E3 deletion was usually performed to provide the necessary space for the insertion of foreign genes of a limited size. The E3 region is non-essential for virus growth in tissue culture, so that vectors deleted only in E3 region could be propagated in non-complementing cells. As E1 region is essential for the virus growth, E1-deleted vectors could only be propagated in complementing cells, such as human 293 cells (ATCC CRL 1573), a human embryonic kidney cell line containing the E1 region of human Ad5 DNA. [0010] One of critical issues in the development of safe viral vectors is to prevent the generation of replication-competent virus during vector production in a packaging cell line or during the gene therapy treatment. This may happen as a result of a recombination event between the genome of the vector and that of the packaging cells, or of the vector and the wild-type virus present in the recipient cells of the patient or introduced as a contaminant in the process of producing the recombinant virus. On occasion, a recombination event could generate a replication-competent virus carrying the transgene, which virus might spread to the environment. Even though recombination events are rare for E1-deleted adenovirus vectors, their in vivo replication and the ensuing risks could not be completely prevented, and generation of replication-competent adenovirus was demonstrated during the preparation of viral stocks. Another danger is the loss of replication deficiency (and the return to a phenotypic state of multiplication) through complementation in trans in some cells which produce proteins capable of replacing proteins encoded by the deleted regions of the viral genome. This was demonstrated for E1-deleted adenoviruses. [0011] Attempts to improve the safety and cloning capacity of adenovirus vectors resulted in development of a new generation of multiply deficient adenovirus vectors (also referred to as second generation or multiply deleted vectors). Additionally to deletions in E1 and/or E3 coding regions, these vectors are also deleted in other regions of the viral genome essential for virus replication, such as early regions E2 and/or E4 (see, for example, WO 95/34671; U.S. Pat. No. 5,700,470; WO 94/28152) or late regions L1-L5 (see, for example, WO 95/02697). Other known approaches to improve the safety of adenovirus vectors include, for example, relocation of protein IX gene in E1-deleted adenovirus (U.S. Pat. No. 5,707,618)- and inactivation of the gene IVa2 in a multiply deleted adenovirus (WO 96/10088). All second generation adenovirus vectors are replication-deficient and require complementing cell lines for their propagation, to restore in trans the deleted or inactivated functions of the viral genome. More importantly, such vectors show an improved resistance to recombination when propagated in complementing cell lines or transferred into recipient cells of a patient, making recombination events virtually nonexistent and improving the safety of gene therapy treatments. [0012] Since their development in the early '80s, adenovirus vectors (AdVs) have been widely used in gene transfer experiments for vaccination (reviewed in: Randrianarison-Jewtoukoff et al., Biologicals, 23, 145-157 (1995)) and in gene therapy (reviewed in: Kovesdi et al., Curr. Opin. in Biotech., 8, 583-589 (1997); Hitt et al., in: The Development of Human Gene Therapy, Cold Spring Harbor Laboratory Press, pp 60-86 (1999)). However, recent developments in the area of adenoviral vectors, such as the increase of insert size, the prolongation and the regulation of transgene expression, as well as the modulation of AdV tropism have further expanded their applications. In particular, adenoviral vectors are now considered as one of the most powerful tools for functional genomics (reviewed in: Oualikene and Massie, in: Cell Engineering, vol. 2, Kluwer Publisher, pp 80-154 (2000); Wang et al., Drug Discov. Today, 5, 10-16 (2000)). Cloning and expressing numerous genes allows the generation of protein libraries useful for various applications, such as signal transduction studies or screening antisense DNA constructs. Such applications of AdVs require a cloning system in which generation and selection of recombinant mutants can be easily performed. An ideal method for the construction of AdV libraries would ensure that i) very large number of clones are generated following transfection of permissive cells, and ii) only recombinant viruses are selected. However, at present the construction of AdVs remains a cumbersome and lengthy process that is not readily amenable to the generation of large collection of clones. [0013] Among the wide variety of methods used for the construction of recombinant AdV, several allow the generation of recombinant viruses without any background of parental genome (Ghosh-Choudhury et al., Gene, 50, 161-171 (1986); Bett et al., Proc. Natl. Acad. Sci. USA, 91, 8801-8806 (1994); Ketner et al., Proc. Natl. Acad. Sci. USA, 91, 6186-6190 (1994); Chartier et al., Escherichia coli J. Virol., 70, 4805-4810 (1996); Crouzet et al., Proc. Natl. Acad. Sci USA, 94, 1414-1419, (1997); He et al., Proc. Natl. Acad. Sci. USA, 95, 2509-2514 (1998); Mizuguchi et al., Hum. Gene Ther., 9, 2577-2583 (1998)). However, for all of these methods the number of viral clones generated is, at best, lower than 50 per .mu.g of viral DNA. Only one method using the viral DNA-protein complex (DNA-TPC), which enhances the number of viral clones by up to 100-fold, was shown to provide large number of clones, albeit without selection for the recombinant ones (Miyake et al., Proc. Natl. Acad. Sci. USA, 93, 1320-1324 (1996)). This method relies on in vivo recombination in 293 cells of the viral genome co-transfected with a transfer vector harboring enough homologous sequences as well as cis-acting elements, such the left ITR which contains the origin of replication and the packaging region. Thus, even though it is currently possible to generate several thousand of viruses per .mu.g of viral DNA, only a fraction of those will be recombinant. [0014] To minimize the work involved in the screening process, reporter genes such as E. coli LacZ (Schaack et al., J. Virol., 69, 3920-3923 (1995)) or the green/blue fluorescent proteins (GFP/BFP) from A. Victoria (Massie et al., Cytotechnology, 28, 53-64 (1998)) can be used either in the viral genome as negative screen, or in the transfer vector as positive screen. Although useful, this approach still suffer from the intrinsic limitation that, in a library of several thousand of clones, an even larger number of parental viruses would have to be screened against, a process which is fairly time consuming. Furthermore, recombinant AdV are sometimes at growth disadvantage relative to the parental virus and these clones might be more difficult to isolate in a library, unless recombinant viruses are positively selected for growth. [0015] Thus far, a positive selection system compatible with the generation of very large number of AdV clones has not yet been developed. One possible approach to do so would be to ectopically re-express an essential gene of adenovirus (which gene has been deleted at its native location) in such a way that only viral genomes that incorporated this gene would be able to grow in the selective environment (a positive selection). The present invention provides such a novel system for cloning DNA sequences in AdVs using the adenovirus protease as an example of the essential gene which can be used for the positive selection. SUMMARY OF THE INVENTION [0016] The present invention provides an adenovirus vector/packaging cell line system in which the vector replication is blocked by deletion of a single gene, not a viral transcriptional region, which deletion does not interfere with any other viral functions. The deleted gene is the gene of the adenovirus protease. The protease encoded by the deleted gene is expressed in a complementing (packaging) cell line through a regulatable expression cassette which induces no toxic effects in the cells, thus making the generation and production of the vector easier and efficient. As the deleted gene is highly specific of adenovirus, no complementation of the gene in transduced cells is to be expected, which increases the safety and suitability of the protease gene deleted vectors for gene transfer purposes. [0017] When additionally deleted for E1 region of adenoviral genome, the vectors of the invention are blocked for replication, but are capable of a single round of replication if deleted only for the protease gene. The latter feature permits an enhanced expression of the transgene in transduced cells, which may be of importance in some applications, for example to achieve localized enhanced expressions of transgenes (in situ tumor therapy) or efficient vaccinations without boosting. [0018] In a preferred embodiment, the invention allows positive selection of E1-deleted, protease-deleted recombinant adenovirus vectors comprising an exogenous gene or an expressible piece of exogenous DNA, by providing an expression cassette comprising the protease gene and the exogenous gene or DNA under control of a suitable promoter, which may be a regulatable (e.g., inducible) promoter, inserted in place of E1 region in a shuttle vector. In another embodiment, the exogenous gene or expressible exogenous DNA is put into a separate expression cassette, under control of a suitable promoter. In vivo recombination of the shuttle vector with a protease-deleted adenoviral genome in suitable non-complementing cells generates viable recombinants only when rescuing the protease cloned in E1 region. Non-recombinant viral genomes are not able to grow due to the deletion of the protease gene, ensuring that only recombinant viral plaques are generated. This positive selection ensures generation of a large number of high purity recombinant adenovirus vectors and allows generation of adenovirus-based expression libraries with diversity exceeding 10.sup.6 clones. [0019] Consequently, it is an object of the present invention to provide novel cell lines capable of complementing in trans an adenovirus mutant deleted for the protease gene, which cell lines contain DNA expressing the adenovirus protease. [0020] It is a further object of the present invention to provide a method for producing novel cell lines capable of complementing in trans an adenovirus mutant deleted for the protease gene, which cell lines contain DNA expressing the adenovirus protease. Continue reading about Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease... Full patent description for Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease or other areas of interest. ### Previous Patent Application: Prevention and treatment of amyloid-associated disorders Next Patent Application: Infectious, chimeric hepatitis c virus, methods of producing the same and methods of use thereof Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease patent info. IP-related news and info Results in 0.35437 seconds Other interesting Feshpatents.com categories: Accenture , Agouron Pharmaceuticals , Amgen , AT&T , Bausch & Lomb , Callaway Golf 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
