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Compositions and methods for increasing packaging and yields of recombinant adenoviruses using multiple packaging signalsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Compositions and methods for increasing packaging and yields of recombinant adenoviruses using multiple packaging signals description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070134203, Compositions and methods for increasing packaging and yields of recombinant adenoviruses using multiple packaging signals. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] The invention relates generally to the field of adenoviral vectors useful in delivering genes and methods of producing same. [0003] Adenoviruses have been described as useful viral vectors for delivery of therapeutic genes into selected host cells. Due to interest in adenoviruses as delivery vehicles, several groups have investigated the mechanisms that allow selective packaging of the adenovirus (Ad) genome into viral capsids. [0004] Within the left end of the Ad genome, a cis-acting packaging domain has been identified [M. Grable and P. Hearing, J. Virol. 64:2047-2056 (May 1990)]. Mutants of Ad serotype 5 (Ad5) lacking this region are nonviable but can be rescued by insertion of the left-terminal 355 nt at the right end of the viral genome. The Ad5 packaging domain has been found to function in an inverted orientation, and can be moved within several hundred base pairs from its original location without a reduction in virus yield [P. Hearing et al, J. Virol., 61:2555-2558 (August 1987)]. [0005] The Ad5 packaging domain consists of at least seven elements which are functionally redundant. Four of the first five elements contain an AT-rich repeated sequence motif termed the A repeat. The fifth element does not contain any obvious primary sequence homology to the A repeat aside from the fact that it is also AT rich. With reference to the published sequences of Ad5, A repeat I is located within nt 240-248; A repeat II is located within nt 260-268; A repeat III is located within nt 302-311; A repeat IV is located within nt 313-321; A repeat V is located within nt 337-346; A repeat VI is located within nt 363 and 368 of Ad5; A repeat VII is located within nt 370-375 of Ad5 [M. Grable and P. Hearing, J. Virol, 66(2):723-731 (Feb. 1992)]. The literature reports efforts to determine the minimum portion of the packaging domain required to package adenovirus DNA into virions. This approach is consistent with on-going efforts to maximize safety of the vectors, as well as to provide adequate space in the viral genome for heterologous gene sequences which are delivered by the adenoviral vectors. [0006] The art continues to search for methods of optimizing production and yield of adenoviral vectors. SUMMARY OF THE INVENTION [0007] The present invention provides compositions and methods useful for efficiently packaging recombinant adenoviruses and producing high yields thereof. The invention involves engineering recombinant adenoviruses to contain multiple functional adenoviral packaging domains. Suitably, these vectors contain at least five of the "A" repeat elements of the adenoviral packaging domains, in duplicate. Most preferably, the vectors contain at least one intact adenoviral packaging domain and a second adenoviral packaging domain containing at least five "A" repeat elements. [0008] Thus, in one aspect, the invention provides (a) an adenovirus 5' inverted terminal repeat, (b) a first adenovirus packaging domain, (c) a second adenovirus packaging domain; (d) a selected transgene under the control of regulatory sequences directing expression of the transgene, and (3) a 3' inverted terminal repeat. Suitably, the second packaging domain is located 5' to the native E1 region and the selected transgene. [0009] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant adenovirus of the invention and a physiologically compatible carrier. [0010] In still another aspect, the invention provides a method of delivering a transgene to a selected host cell by infecting said cell with a recombinant adenovirus of the invention. [0011] In a further aspect, the invention provides a method of increasing the packaging and yield of a selected recombinant adenovirus. The method involves engineering the selected recombinant adenovirus vector to contain at least two adenoviral packaging domains. [0012] In yet a further aspect, the invention provides a method of producing a recombinant adenovirus which lacks functional adenoviral early, intermediate and late genes. The method involves co-culturing in a host cell (a) a recombinant adenoviral plasmid comprising multiple adenoviral packaging domains and a selected transgene, said plasmid lacking functional adenoviral early, intermediate and late genes; and (b) a helper virus. The rAd plasmids and helper virus, together with the host cell, provide sufficient adenoviral gene functions to permit packaging of the recombinant adenoviral plasmid into an adenoviral capsid. [0013] Other aspects and advantages of the invention will be readily apparent from a review of the following detailed description of the invention. DETAILED DESCRIPTION OF THE INVENTION [0014] The present invention provides an adenoviral vector which comprises at least two adenoviral packaging domains, as defined herein. These vectors are characterized by enhanced packaging ability and yield, as compared to viral vectors containing native packaging signals. Thus, the invention provides compositions and methods which have significant advantages in the production of recombinant adenoviral vectors. I. Recombinant Adenovirus Vectors [0015] The recombinant adenoviral vectors of the invention are composed of, at a minimum, adenovirus 5' inverted terminal repeat sequences (ITRs), a first adenovirus packaging domain, a second adenovirus packaging domain, a selected transgene, and an adenovirus 3' ITR. As used herein, the term vector includes, without limitation, any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. [0016] The DNA sequences providing the adenoviral elements and encoding the adenovirus genes useful in this invention may be selected from among any known adenovirus type, including the presently identified 41 human types [see, e.g., Horwitz, cited above]. Similarly, adenoviruses known to infect other animals may supply the gene sequences. The selection of the adenovirus type for each adenoviral element or gene sequence does not limit this invention. The sequences for a number of adenovirus (Ad) serotypes, including that of Ad serotype 5, are available from the GenBank database. [See, e.g., GenBank, accession numbers NC-001406 (human Ad5, complete genome); NC-001460.1 (complete genome human Ad 12); NC-001405 (complete genome human Ad2); AF0865670 (complete cds, human Ad15); AF086571.1 (complete cds, human Ad9); AF086570 (complete cds, human Ad8); AF086569 (complete cds, human Ad37); AF086568 (complete cds, human Ad19), among others]. A variety of adenovirus strains are available from the American Type Culture Collection, Manassas, Va., or are available by request from a variety of commercial and institutional sources. The adenovirus sequences used in the vectors of the invention may be derived from one or more wild-type adenoviruses or mutant adenoviruses. [0017] A. Adenoviral ITRs [0018] The 5' and 3' adenoviral inverted terminal repeats (ITRs) are located at the extreme 5' and extreme 3' ends of the adenoviral genome, respectively. The 5' and 3' ITRs are similarly located at or near the extreme termini of the recombinant adenoviral vectors of the invention. These ITRs may be obtained from any of the adenoviral serotypes described above, or may be artificial sequences, e.g., synthetic or mutated sequences that have the same or equivalent packaging functions as native ITRs. [0019] B. Adenovirus Packaging Domain [0020] As used herein, the term "adenovirus packaging domain" refers to the nucleic acid sequences of the adenoviral repeat elements required to package an adenoviral vector into a capsid protein. Although the packaging domain of the Ad5 is among the best studied of the human serotypes [see, Grable and Hearing (1992), cited above], this invention is not so limited. The repeat sequences of other adenoviral serotypes may be readily substituted in the present invention. Further, one of skill in the art may substitute functional fragments of one or more of the A repeat sequences. Additionally, the repeat elements and/or their functional fragments may be produced synthetically using conventional methods. [0021] Further, in addition to the specific A repeats which have been identified, additional sequences may be included in the adenoviral packaging domains used in the vectors of the invention. For convenience, one may utilize an intact or partial native adenoviral packaging domain which, in addition to containing the A repeat elements, further comprises adenovirus E1a enhancer sequences. Thus, the adenovirus sequences of the packaging domain may extend 3' and/or 5' beyond the functional repeat elements. For example, one suitable adenovirus packaging domain may contain nt 218-354 of Ad5 which encompasses repeats I-V. In another example, a suitable adenovirus packaging domain may contain nt 186-371 which encompasses repeats I-VI and contains a partial repeat VII. Alternatively, the adenoviral packaging domains may further contain other sequences which serve to separate the individual repeat elements and/or the 5' ITRs from the repeats of the adenoviral packaging domain and/or which serve to separate the multiple adenoviral packaging domains. For example, where used to separate individual repeat elements, such sequences may be from 5 bp to 200 bp, 10 bp to 100 bp, 20 bp to 60 bp, or 30 bp to 50 bp in length. However, the length of these sequences may be readily adjusted as needed for convenience. These sequences may be viral in nature, or may obtained from other recombinant or synthetic means, and do not significantly impair the packaging function of the adenoviral packaging domain. Continue reading about Compositions and methods for increasing packaging and yields of recombinant adenoviruses using multiple packaging signals... 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