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  • 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
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/10011—Adenoviridae
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10351—Methods of production or purification of viral material
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

• the present invention relates generally to compositions and methods useful in producing recombinant viruses, and particularly, those methods which require the use of a helper virus. More particularly, the invention provides compositions and methods useful in obtaining a recombinant viral particle in a form which is readily separated from cultures in which helper virus was used in the production.
• cre-loxP system has been the primary system available to generate helper dependent adenovirus vectors with diminished helper virus [Parks et al, Proc. Natl. Acad. Sci USA, 93: 13565-13570 (1996); Parks, J. Virol, 71:3293-3298 (1997)].
• the present invention provides compositions and methods which permit production of recombinant viral vectors which are readily purified from helper viruses.
• the method uses a recombinant helper virus which has been designed to contain at least one site for a rare-cutting restriction enzyme, a viral vector which is helper-dependent for packaging into a virus, and a recombinant host cell which expresses the rare-cutting restriction enzyme.
• the rare- cutting enzyme is I-Scel.
• the method involves transfecting or infecting the host cell with the helper virus and viral vector and incubating the cell under conditions which permit packaging of the viral vector.
• the rare-cutting restriction enzyme expressed by the host cell digests the helper virus, permitting ready separation of the digested fragments of the helper virus from the packaged recombinant virus.
• the restriction enzyme is I-Scel and the viral vector is an adenovirus.
• the present invention provides a method for producing helper-dependent virus which involves the following steps.
• a host cell capable of expressing a rare-cutting restriction enzyme e.g., I-Scel
• the host cell is transfected with a recombinant viral vector comprising a minigene containing a transgene encoding a selected protein and regulatory sequences which control expression of said protein.
• the host cell is infected with a recombinant helper virus engineered to contain at least one, and preferably multiple restriction sites for the rare-cutting restriction enzyme, e.g., I-Scel.
• the host cell with the recombinant viral vector and helper virus is then cultured under conditions which permit packaging of the recombinant viral vector in a viral particle, wherein the helper virus and the host cell provide to the recombinant adenovirus vector the necessary viral genes for viral packaging.
• the present invention provides a recombinant virus produced by the method of the invention, which is substantially purified from helper virus.
• the invention provides a stable mammalian cell line which expresses a rare-cutting restriction enzyme, e.g., I-Scel.
• the invention provides a recombinant helper virus useful in helper-dependent production of a recombinant virus vector, wherein said helper virus is engineered to contain a site for a rare-cutting restriction enzyme downstream of the packaging signal.
• the invention provides a method for the helper- dependent production of a recombinant virus which involves transfecting a host cell with a recombinant viral vector comprising a minigene containing a transgene encoding a selected protein and regulatory sequences which control expression of said protein.
• the host cell is further infected with a recombinant helper virus engineered to contain a site for a rare-cutting enzyme, e.g., I-Scel, and the corresponding restriction enzyme, e.g., I-Scel, is delivered to the host cell.
• the host cell is then cultured under conditions which permit packaging of the recombinant virus vector in a viral particle.
• the helper virus and the host cell provide to the recombinant viral vector the necessary viral genes for viral packaging and the restriction enzyme cleaves the recombinant helper virus following generation of the recombinant viral vector.
• the invention provides a cell lysate comprising a recombinant virus which is substantially free of helper virus.
• Fig. 1A is a schematic illustration of the generation of recombinant adenoviruses using I-Scel expressed from the human 293 cell line.
• Fig. IB is a schematic illustration of the generation of recombinant adenoviruses using recombinant AAV vectors to deliver the functional I-Scel enzymes
• Fig. 2 is a detailed restriction map for a recombinant adenovirus helper virus of the invention, Ad-3I.
• Ad-3I a recombinant adenovirus helper virus of the invention
• the CMV- ⁇ l -antitrypsin cassette was located right after the 3' end of adenovirus genome.
• Fig. 3 is a line graph providing a time course of gene expression from Ad-3I in the presence of I-Scel enzyme in human 293 cells.
• Ad-3I MOI 10
• Ad-3I MOI 10
• 293 cells 293-I-SceI cell line or 293 cells along with rAAV-I-Scel at MOI 10.
• the amount of human ⁇ l -antitrypsin in media was measured by ELISA at the various times indicated in the graph.
• Fig. 4 is a bar chart illustrating that 293 I-Scel cell lines enhanced helper dependent adenovirus production.
• Fig. 5 is a bar graph illustrating the performance of an I-Scel cell line of the invention, as determined by helper virus at 48 hours post-infection shown as the ⁇ l- antitrypsin secreted into the media.
• the digested helper virus is not able to express ⁇ l -antitrypsin.
• the same amount of Ad- ⁇ -LacZ vector and Ad-3I mix were used to infect the same amount of 293, 293-I-SceI(a), 293-I-SceI(b) cells.
• the viruses were harvested 48 hours post-infection.
• the lacZ-forming unit (LFU) was determined by X-Gal staining.
• the present invention provides compositions and methods which permit production of recombinant viral vectors which are readily separated from helper viruses.
• the invention is particularly suited for production of viral vectors which are dependent upon helper viruses for packaging and/or encapsidation into a recombinant virus.
• the invention uses a recombinant helper virus which has been designed to contain at least one, and preferably, multiple sites for a selected rare- cutting restriction enzyme and a recombinant host cell which is capable of expressing the rare-cutting restriction enzyme.
• a helper virus is any virus which, in conjunction with the selected host cell, provides the necessary viral products to permit packaging and/or encapsidation of the selected viral vector into an infectious recombinant virus.
• the host cell may be a cell line stably or transiently expressing the rare- cutting restriction enzyme.
• the method involves delivering the helper virus and viral vector to a host cell which expresses the restriction enzyme (or is modified to express the enzyme) and culturing the cell under conditions which permit encapsidation of the viral vector. Thereafter, the restriction enzyme expressed by the host cell digests the helper virus, permitting ready separation of the digested fragments of the helper virus from the packaged recombinant virus.
• a rare-cutting restriction enzyme is a restriction enzyme which is not naturally present in the selected host cell and/or selected viral vector genome or which recognizes a site which is present sufficiently infrequently in the genomes of the species of the host or virus, that the site (and/or enzyme) is unlikely to occur naturally in the host cell or viral vector.
• a suitable rare-cutting enzyme for use in the present invention is the restriction enzyme I-Scel.
• the I-Scel enzyme is an endonuclease encoded by the group I intron of S cerevisiae mitochondria [L. Colleaux et al, Proc. Natl. Acad. Sci. USA, 85:6022-6026 (1988), which has high specificity for an 18 bp nonpalindromic nucleotide sequence [I-Scel site: SEQ ID NO: 1 : 5'-tagggataa/cagggtaat].
• a common restriction endonuclease generally recognizes a short stretch of nucleotides of 4 to 8 base pairs; thus, there would be about one million such sites in one human genome.
• I-Scel site occurs randomly only once in every 20 human genomes.
• the rarity of I-Scel sites has been partially confirmed by the fact that there are no I-Scel sites in the genomes of many organisms, including viruses, bacteria and yeast. Based on the information provided herein, one of skill in the art can readily substitute other suitable rare-cutting restriction enzymes for I-Scel for use in the invention.
• rare-cutting restriction enzymes may be selected, for example, from among various restriction enzymes which are native to non-mammalian animals, plants, yeast, fungi, and/or insects, which restriction enzymes are not native to mammalian species.
• Examples of rare-cutting restriction enzymes, in addition to I- Scel include, Pspl, I-Ceul, and the like.
• Other suitable rare-cutting restriction enzymes may be selected from among those which recognized sites which are at least about 12 to about 40 nucleotides in length, preferably about 14 to about 20 nucleotides in length, and most preferably, at least about 18 nucleotides in length.
• restriction enzymes are available from a variety of commercial sources, including, e.g., New England BioLabs, Promega, and Boehringer Mannheim. Alternatively, these enzymes or their coding sequences may be produced synthetically or using recombinant technology. For example, the I-Scel enzyme may be purchased from commercial sources
• the sequence of the enzyme may be produced by conventional chemical synthesis. [See, e.g., G. Barony and R.B. Merrifield, The Peptides: Analysis, Synthesis & Biology, Academic Press, pp. 3-285 (1980)].
• the native coding sequence for this enzyme is altered to optimize expression in mammalian cells, which are the preferred host cells. Techniques for optimizing expression, e.g., by altering preference codons, are well known to those of skill in the art.
• sequences for the I-Scel site and, other selected rare-cutting restriction enzyme sites may be produced synthetically, recombinantly, or obtained using other suitable techniques. See, e.g., Barony and Merrifield, cited above; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York.
• the invention provides host cells which are useful in the production of helper- dependent viruses.
• the invention provides cell lines which stably express a rare-cutting restriction enzyme.
• the sequences encoding the rare-cutting restriction enzyme are delivered in trans to a selected host cell via a suitable vector or other nucleic acid molecule.
• I-Scel enzyme For convenience throughout this specification, reference will be made to the I-Scel enzyme. However, it will be readily understood that another rare-cutting restriction enzyme and/or its restriction enzyme site, as defined herein, may be substituted.
• a cell line of the invention may be constructed by providing the selected host cell line with a nucleic acid molecule encoding a rare-cutting restriction enzyme or a functional fragment thereof operably linked to regulatory sequences which control expression thereof using conventional techniques.
• "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
• Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
• polyA polyadenylation
• a great number of expression control sequences, native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
• the nucleic acid molecule encoding the rare-cutting restriction enzyme e.g., I-Scel
• a nuclear localization signal which targets the I-Scel sequences to the nucleus.
• Suitable nuclear localization signals are known to those of skill in the art and are not a limitation of the present invention.
• the host cell stably expresses the rare-cutting restriction enzyme, e.g., I-Scel, under the control of a constitutive promoter.
• the rare-cutting restriction enzyme e.g., I-Scel
• promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl ⁇ promoter [Invitrogen].
• RSV Rous sarcoma virus
• CMV cytomegalovirus
• PGK phosphoglycerol kinase
• the rare-cutting restriction enzyme e.g., I- Scel
• Inducible promoters are regulated by exogenously supplied compounds, including, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)- inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al, Proc.
• the cell line selected for transformation with the nucleic acid molecule encoding the rare-cutting restriction enzyme, e.g., I-Scel, and, optionally, expression control sequences therefor is a mammalian cell line, including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, NERO, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat and hamster.
• cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, NERO, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells
• Preferred cells include human cells, and most preferably, cells which express adenovirus El functions, e.g., 293 cells.
• ATCC American Type Culture Collection
• other cell lines may be readily obtained from the American Type Culture Collection (ATCC) or a variety of commercial and academic sources.
• the method of the invention is performed by delivery of sequences encoding the rare-cutting restriction enzyme, e.g., I-Scel, or a functional fragment thereof to a selected host cell in trans.
• the nucleic acid molecule carrying the rare-cutting restriction enzyme (e.g, I-Scel) coding sequences and expression control sequences may be in any form which transfers these components to the host cell and permits expression of the restriction enzyme, preferably in the cell nucleus.
• the selected host cell is preferably a human cell line, e.g., 293.
• other suitable host cells may be readily selected from among those known in the art. See discussion of host cells above.
• the nucleic acid molecule carrying the sequences encoding the rare- cutting restriction enzyme (e.g., I-Scel), as well as the sequences which regulate expression thereof, are provided to the host cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. See, for instance, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. Most suitably, these sequences are contained within a vector.
• a "vector” includes, without limitation, any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc..
• the restriction enzyme (e.g., I-Scel) coding sequences are delivered via a viral vector, and most preferably, via a recombinant, infectious virus.
• Selection of the enzyme delivery virus is not a limitation on the present invention.
• Suitable recombinant enzyme (e.g., I-Scel) delivery viruses may be readily engineered utilizing such viruses as adeno-associated viruses (AAV), retroviruses, adenoviruses, hybrid adeno-AAV viruses, lentiviruses, baculovirus, herpes virus, and pox viruses, among others.
• the enzyme delivery virus is a recombinant AAV (rAAV) containing the enzyme coding sequences operably linked to suitable expression control sequences, which direct expression of the enzyme (e.g., I-Scel) in the host cell and target the nucleus.
• rAAV recombinant AAV
• suitable expression control sequences which direct expression of the enzyme (e.g., I-Scel) in the host cell and target the nucleus.
• I-Scel-rAAV and other delivery virus constructs of the invention are prepared using the rare-cutting restriction enzyme sequences, obtained as described herein, and using known methods. For example, methods for producing rAAV vectors have been described. [See, W. Xiao et al, J. Virol, 72: 10222-10226 (1998); US Patent No. 5,658,776; US Patent No. 5,622,856, among others].
• an I-Scel-rAAV of the invention employs the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences [see, e.g, B.j. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)] flanking the sequences encoding I-Scel and directing expression thereof.
• ITR sequences are about 145 bp in length.
• substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art.
• An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences.
• the AAV ITR sequences may be obtained from any known AAV, including presently identified human AAV types.
• AAVs known to infect other animals may also provide these ITRs employed in the molecules or constructs of this invention.
• the ITRs may be provided by AAV type 1, AAV type 2, AAV type 3, AAV type A, AAV type 5, parvovirus type HI, MVM, LuIII, or by any other parvovirus or AAV serotype.
• a variety of AAV strains are available from the American Type Culture Collection or are available by request from a variety of commercial and institutional sources.
• an AAV-2 is used for convenience.
• the selection of the species and serotype of AAV that provides these sequences is within the skill of the artisan according to the teachings of this application and does not limit the following invention.
• the enzyme delivery virus contains the restriction enzyme nucleic acid sequences, a nuclear localization signal, and conventional regulatory elements necessary to drive expression of the enzyme in a host cell transfected (infected) with this enzyme delivery virus.
• expression control elements include promoters, including both constitutive and inducible promoters, as are described above, poly A sequences, and the like.
• Other heterologous nucleic acid sequences optionally present in this enzyme delivery virus include sequences providing signals required for efficient polyadenylation of the RNA transcript, and introns with functional splice donor and acceptor sites.
• a common poly-A sequence which is employed in the enzyme delivery viruses useful in this invention is that derived from the papovavirus SV-40.
• the poly-A sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence.
• An enzyme delivery virus useful in the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
• One possible intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18-3.26 and 16.17-16.27].
• the enzyme delivery virus may contain a selectable marker or reporter sequences, such as sequences encoding hygromycin or purimycin, among others. See the discussion of reporter sequences below.
• the I-Scel-rAAV may be delivered to the selected host cells at a multiplicity of infection (MOI) of about 5 to about 200 rAAV genome particles, and preferably at an MOI of 10 to 100 rAAV genome particles. Suitable MOI for other selected enzyme delivery viruses may be in this range, or may be adjusted as desired by one of skill in the art.
• MOI multiplicity of infection
• the I-Scel is delivered by a vector which lacks the ability to infect host cells
• the vector is delivered to the host cells in an amount of about 5 ⁇ g to about 100 ⁇ g DNA by any suitable means known to those of skill in the art.
• the enzyme delivery vehicle may be provided to the host cells at any time prior to cell lysis, including prior to delivery of the vector (e.g., by infection or transfection), prior to delivery of the helper virus sequences (e.g., infection with the helper virus), or after delivery of either or both of these components to the host cell.
• the enzyme delivery vehicle constitutively expresses the restriction enzyme (e.g., I-Scel)
• the enzyme delivery vehicle inducibly expresses the restriction enzyme
• the timing of delivery of the enzyme may not be critical. However, the selection of promoters, and the determination of timing of delivery of an enzyme delivery virus (or other nucleic acid molecule) to the host cell may be made by one of skill in the art in view of the information provided herein.
• compositions and methods of the present invention are particularly well suited for packaging of a recombinant viral vector which lacks sufficient viral genes to permit encapsidation in a capsid in the absence of a helper virus and host cell. Together, the helper virus and host cell provide the gene functions necessary to encapsidate the recombinant virus.
• This present invention may be utilized for any viral vector which is dependent upon a helper virus for packaging.
• viral vectors may include, without limitation, recombinant adeno-associated vectors (rAAV), recombinant adenovirus vectors, hybrid adenovirus/AAV vectors, retroviruses, and lentiviruses. Selection of a suitable viral vector is not a limitation of the present invention.
• the method of the invention is particularly well suited for packaging of adenoviral vectors.
• the adeno viral vector contains only minimal adenovirus sequences.
• the recombinant adenovirus vector referred to herein as pAd ⁇ is described below. Methods of producing these recombinant adenoviral vectors are known in the art. See, e.g., International Patent Publication No. WO96/13597. A. The Adenovirus Sequences ofpAdA
• the adenovirus nucleic acid sequences of the pAd ⁇ vector provide the minimum adenovirus sequences which enable a viral particle to be produced with the assistance of a helper virus and, optionally, a packaging cell line. These sequences assist in delivery of a recombinant transgene genome to a target cell by the resulting recombinant virus.
• the DNA sequences of a number of adenovirus types are available from Genbank, including type Ad5 [Genbank Accession No. M73260].
• the adenovirus sequences may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified human types [see, e.g., Horwitz, cited above].
• adenoviruses known to infect other animals may also be employed in the vector constructs of this invention.
• the selection of the adenovirus type is not anticipated to limit the following invention.
• a variety of adenovirus strains are available from the American Type Culture Collection, Manassas, Virginia, or available by request from a variety of commercial and institutional sources.
• an adenovirus type 5 (Ad5) is used for convenience.
• pAd ⁇ vectors based on different human adenovirus serotypes. It is anticipated that a library of such plasmids and the resulting Ad ⁇ viruses would be useful in a therapeutic regimen to evade cellular, and possibly humoral, immunity, and lengthen the duration of transgene expression, as well as improve the success of repeat therapeutic treatments. Additionally the use of various serotypes is anticipated to produce recombinant viruses with different tissue targeting specificities.
• the pAd ⁇ vector lacks nucleic acid sequences encoding all functional adenoviral genes.
• Such functional genes include El, E2, E3, E4, the intermediate genes (IVa and IX) and late genes (LI, L2, L3, L4, L5).
• the adenovirus sequences employed are the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5' packaging/enhancer domain that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the El promoter. These sequences are necessary for replication and virion encapsidation. See, e.g., P. Hearing et al, J. Virol.. 6J_(8):2555-2558 (1987); M. Grable and P. Hearing, J. Virol. 64(5): 2047-2056 (1990); and M. Grable and P. Hearing, J. Virol. 66(2):723-731 (1992).
• the entire adenovirus 5' sequence containing the 5' ITR and packaging/enhancer region can be employed as the 5' adenovirus sequence in the pAd ⁇ vector.
• This left terminal (5') sequence of the Ad5 genome useful in this invention spans bp 1 to about 360 of the conventional adenovirus genome, also referred to as map units 0-1 of the viral genome.
• This sequence includes the 5' ITR and the packaging/enhancer domain.
• this adenovirus 5' region is employed in the vector in its native, unmodified form. However, some modifications including deletions, substitutions and additions to this sequence which do not adversely effect its biological function may be acceptable. See, e.g., International Patent Publication No.
• the 3' adenovirus sequences of the vector include the right terminal (3') ITR sequence of the adenoviral genome spanning about bp 35,353 - end of the adenovirus genome, or map units ⁇ 98.4-100. This entire sequence is desirably employed as the 3' sequence of a pAd ⁇ vector.
• the native adenovirus 3' region is employed in the shuttle vector in unmodified form. However, some modifications to this sequence which do not adversely effect its biological function may be acceptable.
• An exemplary pAd ⁇ vector used in this invention lacks adenovirus sequences encoding functional adenoviral genes.
• the pAd ⁇ vector contains Ad5 5' and 3' cis-elements, as well as the transgene sequences described below.
• these 5' and 3' elements may flank the transgene (e.g., 5' cis- elements, transgene, 3' cis-elements).
• these 5' ITRs and 3' ITRs may be oriented in a head-to-tail configuration, located upstream of the transgene.
• Such a vector may be constructed using conventional genetic engineering techniques, e.g., homologous recombination and the like. See, e.g., US Patent No. 6,001,557.
• adenovirus sequences for use in the pAd ⁇ vectors of this invention. These sequences may include other adenovirus strains, or the above mentioned cis-acting sequences with minor modifications. Further, one of skill in the art will readily appreciate that the method of the invention is useful in overcoming difficulties in separating helper virus from production cultures for other viral vectors lacking gene functions required for packaging. Such other vectors include rAd vectors lacking one or more adenoviral genes selected from El, E2, E4, the intermediate genes and the late genes, as well as non-adenoviral vectors.
• non- adenoviral vectors which require helper functions for production include , without limitation, parvoviruses, including adeno-associated viruses and retroviruses, including, lentiviruses.
• parvoviruses including adeno-associated viruses and retroviruses, including, lentiviruses.
• retroviruses including, lentiviruses.
• One of skill in the art can readily select other suitable systems for use of the present invention.
• the transgene sequence of the recombinant vector and the virus resulting from the method of the invention is a nucleic acid sequence, heterologous to the virus sequence, which encodes a polypeptide, protein, or other product, of interest.
• the transgene is operatively linked to regulatory components in a manner which permits transgene transcription.
• transgene sequence will depend upon the use to which the resulting virus will be put.
• one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal.
• reporter sequences include without limitation, DNA sequences encoding ⁇ - lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.
• sequences when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of helper virus is detected by assays for beta-galactosidase activity. Where the transgene is luciferase, the helper virus may be measured by light production in a luminometer.
• the transgene is a non-marker sequence encoding a product which is useful in biology and medicine, such as proteins, peptides, anti- sense nucleic acids (e.g., RNAs), enzymes, or catalytic RNAs.
• the transgene may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed.
• a preferred type of transgene sequence encodes a therapeutic protein or polypeptide which is expressed in a host cell.
• the invention further includes using multiple transgenes, e.g., to correct or ameliorate a gene defect caused by a multi-subunit protein.
• a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein.
• a cell is infected with the recombinant virus containing each of the different subunits.
• different subunits of a protein may be encoded by the same transgene.
• a single transgene includes the DNA encoding each of the subunits, with the DNA for each subunit separated by an internal ribozyme entry site (IRES).
• IRES internal ribozyme entry site
• the selected transgene may encode any product desirable for study.
• the selection of the transgene sequence is not a limitation of this invention.
• Useful products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor ⁇ (TGF ⁇ ), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor ⁇ superfamily, including TGF ⁇ , activins, inhibins, or any of the bone morphogenic proteins
• transgene products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-17, monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors ⁇ and ⁇ , interferons ⁇ , ⁇ , and ⁇ , stem cell factor, flk-2/flt3 ligand.
• TPO thrombopoietin
• IL interleukins
• monocyte chemoattractant protein monocyte chemoattractant protein
• leukemia inhibitory factor granulocyte-macrophage colony stimulating factor
• Fas ligand tumor necrosis factors ⁇ and ⁇
• tumor necrosis factors ⁇ and ⁇ interferons ⁇ , ⁇ , and ⁇
• stem cell factor flk-2/flt
• immunoglobulins IgG, IgM, IgA, IgD and IgE include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules.
• Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.
• complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2 and CD59.
• Still other useful gene products include any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins.
• the invention encompasses receptors for cholesterol regulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and the scavenger receptor.
• the invention also encompasses gene products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors.
• useful gene products include transcription factors such as jun,fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
• transcription factors such as jun,fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box
• genes include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T- protein, a
• Non-naturally occurring polypeptides such as chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions.
• polypeptides such as chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions.
• single-chain engineered immunoglobulins could be useful in certain immunocompromised patients.
• Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which could be used to reduce overexpression of a gene.
• Other suitable transgenes may be readily selected by one of skill in the art. The selection of the transgene is not considered to be a limitation of this invention.
• the vector also includes conventional control elements necessary to drive expression of the transgene in a cell transfected with the viral vector.
• the vector contains a selected promoter which is linked to the transgene and located, with the transgene, between the viral sequences of the vector. Suitable promoters may be readily selected from among constitutive and inducible promoters, such as those discussed herein.
• minigene is flanked by the 5' and 3' cis-acting adenovirus sequences described above.
• minigene may have a size in the range of several hundred base pairs up to about 30 kb due to the absence of adenovirus early and late gene sequences in the vector.
• this viral vector e.g., pAd ⁇
• pAd ⁇ a viral vector
• this viral vector permits a great deal of latitude in the selection of the various components of the minigene, particularly the selected transgene, with regard to size.
• the design of such a minigene can be made by resort to conventional techniques.
• the recombinant viral vector e.g., pAd ⁇
• Delivery can be by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
• transfection is a particularly desirable method of delivering the viral vector (e.g., pAd ⁇ ) to the host cell.
• pAd ⁇ is linearized by a suitable restriction enzyme and added in transfection cocktail.
• transfection is then performed using the calcium-phosphate based techniques described in Cullen, in "Methods in Enzymology", ed. S.L. Berger and A.R. Kimmel, Vol. 152, pp. 684-704, Academic Press, San Diego (1987).
• Other suitable transfection techniques are known and may readily be utilized to deliver the recombinant vector to the host cell.
• the vector when delivering the recombinant vector (e.g., pAd ⁇ ) by transfection, the vector is delivered in an amount from about 5 ⁇ g to about 100 ⁇ g
• DNA DNA, and preferably about 10 to about 50 ⁇ g DNA to about 1 x 10 4 cells to about 1 x 10 13 cells, and preferably about 10 5 cells.
• the relative amounts of vector DNA to host cells may be adjusted, taking into consideration such factors as the selected vector, the delivery method and the host cells selected.
• the present invention utilizes a recombinant helper virus for production of the viral vector, which helper virus has been engineered to contain heterologous restriction sites for the rare-cutting restriction enzyme, e.g., I-Scel.
• helper virus has been engineered to contain heterologous restriction sites for the rare-cutting restriction enzyme, e.g., I-Scel.
• the production of a recombinant virus which utilizes helper virus containing a full complement of required genes results in recombinant virus contaminated by excess production of the helper virus.
• extensive purification of the recombinant virus from the contaminating helper virus is required.
• the present invention provides a way to facilitate purification and reduce contamination by enzymatic digestion of the helper virus by the rare-cutting restriction enzyme (e.g., I-Scel) expressed by the host cell.
• helper viruses of the invention contain at least one, and preferably multiple, rare-cutting restriction enzyme sites, which are located within the genome of the helper virus such that the digested helper virus is composed of fragments which are small enough to be readily distinguishable from the recombinant virus and any nucleic acid used to deliver the restriction enzyme.
• helper virus is utilized in the production of a recombinant virus from a vector which contains insufficient viral genes to package (or encapsidate) the vector. Because of the limited amount of viral sequence present in the viral vector, a helper virus of this invention must, alone or in concert with a packaging cell line, provide the gene sequences necessary for a productive viral infection.
• a suitable helper virus may be readily selected by one of skill in the art, taking into consideration the viral vector to be packaged. For example, an AAV vector may be packaged in either a rAAV or a recombinant adenovirus (Ad) capsid, using a rAAV or Ad helper virus of a selected serotype, as appropriate.
• helper viruses used in the present invention are capable of replication in the selected host cell.
• the helper viruses are replication-competent in the selected host cells and replication-incompetent in other cells, which do not provide the necessary gene functions to permit replication.
• a pAd ⁇ vector is packaged into an adenoviral capsid using helper viruses which contain selected adenovirus gene sequences, and optionally reporter sequences operably linked to expression control sequences.
• the adenovirus sequences forming the helper virus may be obtained from the sources identified above in the discussion of the pAd vector.
• Ad serotypes as helper viruses enables production of recombinant viruses containing the pAd ⁇ vector sequences in a capsid formed by the other serotype adenovirus.
• Use of these different Ad serotype helper viruses may also demonstrate advantages in recombinant virus production, stability and packaging.
• the helper virus supplies the adenovirus sequences necessary for encapsidation of pAd ⁇ , including early genes El, E2, E4, or fragments of a gene which perform the same or substantially the same function as the intact complete gene (i.e., functional fragments), and all remaining late, intermediate, structural and non-structural genes of the adenovirus genome, which are not present in the pAd ⁇ vector or provided by the cell line.
• the helper virus and cell line provide, at a minimum, adenovirus Ela, Elb, E2a, or functional fragments thereof.
• the helper virus and cell line provide, Ela, Elb, E2a, E4, and VAI, or functional fragments of these genes (e.g., E4 ORF6) and the helper virus provides the adenoviral gene IX function.
• the recombinant virus is an adenovirus and, most preferably, an adenovirus which replicates in the selected host cell.
• other suitable viruses e.g., herpesviruses, may be used as helpers.
• the selected helper virus is engineered to contain at least one, and preferably multiple, restriction enzyme sites (e.g., I-Scel). In one embodiment, the helper virus useful in the present invention contains three heterologous restriction enzyme sites.
• helper virus is digested by the rare-cutting restriction enzyme expressed in the cell, resulting in small fragments of helper virus which are readily distinguishable from and purified from the packaged recombinant virus (e.g., Ad ⁇ ) and any remaining unpackaged viral vector (e.g., pAd ⁇ ).
• packaged recombinant virus e.g., Ad ⁇
• pAd ⁇ any remaining unpackaged viral vector
• the helper virus contains a restriction enzyme site (e.g., a I- Sce-I site) located downstream of the packaging sequences, so that the helper virus is disabled for replication and packaging following digestion with the restriction enzyme expressed in the host cell.
• a restriction enzyme site e.g., a I- Sce-I site
• Other restriction enzyme sites may be readily engineered into other suitable sites to disrupt functions which are no longer required following packaging of the viral vector and/or to permit monitoring of helper virus levels, e.g. by expression of a marker (i.e. reporter) sequence, which upon expression produces a detectable signal. Examples of suitable marker sequences are described above.
• a rare-cutting restriction enzyme site (e.g., I- Scel) is placed upstream of the sequences encoding the marker, permitting monitoring of changing levels of intact helper virus by monitoring a decrease in the levels of detectable marker.
• the helper virus is composed of adenovirus 5' sequences, including a packaging signal, a first I-Scel site, a promoter, a second I-Scel site, a reporter gene, a third I-Scel site, and additional adenovirus sequences.
• Other suitable helper viruses may be readily designed and produced using the information provided herein.
• the present invention provides a method of producing helper-dependent viruses in a host cell which stably expresses the selected rare- cutting restriction enzyme (e.g., I-Scel).
• the host cell is transfected with the recombinant viral vector, e.g., pAd ⁇ , and infected with the helper virus using conventional methods. See, generally, Sambrook et al, cited above. See also, the methods described in K.J. Fisher et al, Virol, 217: 11-22 (1996).
• the host cell is then cultured under suitable conditions to permit encapsidation of recombinant viral vector in a first round of amplification.
• the rare-cutting restriction enzyme expression e.g., I-Scel
• the inducing agent is added 6 to 72 hours, and preferably 24 hours, after infection of the cells with the helper virus.
• the host cell is transiently transfected or infected with the sequences encoding the rare-cutting restriction enzyme (e.g., I- Scel).
• the host cell provided with the recombinant viral vector and helper virus as described above, is then cultured in a similar manner to provide recombinant virus in a viral capsid.
• recombinant virus produced by this first round of viral amplification may then be recovered by conventional means.
• the host cells may be harvested and lysed using known methods, e.g., three rounds of freeze (e.g., ethanol, dry ice) - thawing (e.g., 37°C). The cell lysate may then be centrifuged to remove cell debris.
• An advantage of the present invention is that, due to the difference in size between the recovered virus and the fragments into which the helper virus is cleaved by the restriction enzyme (e.g., I-Scel), the recovered virus is readily separated from the helper virus fragments.
• the recovered virus may be subjected to further purification steps. See, Fisher et al, cited above.
• the recovered lysate may be subjected to a second round of amplification, e.g., using the steps and host cells described above in the first round of amplification. This virus is useful for a variety of purposes known in the art.
• the recombinant viruses produced according to the method of the invention are substantially free of helper virus contamination and are useful for a variety of purposes, which are well known to those of skill in the art.
• Ad-3I The helper adenovirus described in this example is termed Ad-3I since it carries three I-Scel sites in its genome.
• I-Scel coding region was synthesized by Anagen Inc. (CA, USA) and contains modified codons to optimize expression in mammalian cells.
• This complete coding sequence for I-Scel is: SEQ ID NO:2: GGGGCGGCCG CAGCCGCCAT ATGAAGAACA TCAAGAAGAA CCAGGTGATG AACCTGGGCC CCAACAGCAA GCTGCTGAAG GAGTACAAGA GCCAGCTGAT CGAGCTGAAC ATCGAGCAGT TCGAGGCCGG CATCGGCCTG ATCCTGGGCG ACGCCTACAT CAGGAGCAGG GACGAGGGCA AGACCTACTG CATGCAGTTC GAGTGGAAGA ACAAGGCCTA CATGGACCAC GTGTGCCTGC TGTACGACCA GTGGGTGCTG AGCCCCCCCCCC ACAAGAAGGA GAGGGTGAAC CACCTGGGCA ACCTGGTGAT CACCTGGGGC GCACCA GGCCTTCAAC AAGCTGGCCA ACCTGTTC AT C
• the pAd-linker-3I was constructed as follows. The synthetic I-Scel oligonucleotide described in A above was inserted into p Ad-linker 1 at Bgl II and Hind III [Fisher et al, cited above]. This plasmid contains an intact adenoviral genome located on a pBR322 backbone. Another I-Scel site is cloned into pCI-AAT ([Xiao et al, J. Virol, 72: 10222-10226 (1998)] following digestion with Xhol.
• Hind Ill-Cla I fragment of pCI-AAT with one I-Scel was then cloned into pAd- linkerl with two I-Scel sites following digestion with EcoRV and Clal, to obtain pAd-linker-3I.
• Ad-3I The helper virus Ad-3I was constructed by homologous recombination between pAd-linker-3I and adenovirus DNA in 293 cells as described elsewhere [Gao et al, J. Virol, 70:8934-8943 (1996)]. The genome organization of Ad-3I is shown in Figure 2.
• the resulting helper virus, Ad-3I contains from 5 No 3': an adenovirus 5' ITR and packaging signal, a first I-Scel site, a CMV promoter, a second I-Scel site between the CMV promoter and the ⁇ l -antitrypsin cD ⁇ A, and a third I- Scel site between ⁇ l -antitrypsin and the other adenovirus early and late genes.
• the Ela and Elb genes are disrupted in this construct, which replicates in the presence of the El functions provided by the cell line.
• the ⁇ l -antitrypsin gene driven by CMV is included in this helper virus to serve as a reporter gene.
• Digestion of the second I-Scel site (located between the CMV promoter and ⁇ l -antitrypsin cD ⁇ A) results in the loss of ⁇ l -antitrypsin gene expression. Additionally, digestion of any of the three I-Scel sites disables Ad- 31 for replication and packaging since the 5' terminal repeat is thereby detached from helper adenovirus genome.
• Ad-3I D ⁇ A The in vitro digestion of D ⁇ A by I-Scel was carried out using commercially available I-Scel (Boehringer Mannheim, Germany) under the conditions recommended by the vendor.
• I-Scel Boehringer Mannheim, Germany
• the purified virus was digested with proteinase K and extracted from CsCl gradient purified adenovirus with phenol-chloroform before ethanol precipitation.
• the digested D ⁇ A was electrophoresed in 0.8% agarose gel. The size of the bands was identified.
• the complete digestion of Ad-3I results in four fragments of sizes of approximately 34 kb, 1.7 kb, l.l kb and 400 bp.
• Example 2 Production of Human Cell Line Expressing Functional I-Scel Enzyme
• the synthesized I-Scel sequence described in Example 1 A was fused at its ⁇ - terminus with a nuclear translocation signal ( ⁇ LS) to ensure that the enzyme will reach the nucleus where adenovirus D ⁇ A replication takes place. This fusion was performed by inserting the I-Scel sequence downstream of a ⁇ LS in a suitable plasmid backbone.
• ⁇ LS nuclear translocation signal
• the ⁇ LS-I-Scel fragment was then removed by digestion with BspHI and ⁇ otI and the resulting fragment was cloned into plasmid, pEF-myc-nuc (purchased from Invitrogen), to get pEF-Scel pEF-myc-nuc contains a strong constitutive promoter derived from the human elongation faction l .
• the 293-I-SceI cell line was constructed by transfecting plasmid pEF-Scel into 293 cells using lipofectAmine (Gibco BRL) selected under 200 ⁇ g/ml of G418. The G418 resistant clones (22) were selected and analysed for I-Scel expression following infection with Ad-3I at MOI 10.
• the Hirt DNA was extracted 36 hours post-infection. Southern analysis was performed using a human ⁇ l- antitrypsin cDNA probe. Fragments of 1.7 kb and 2.8 kb were obtained. The 1.7 kb fragment results from the digestion of I-Scel sites before and after ⁇ 1 -antitrypsin gene in Ad-3I. The results indicated that most G418 positive clones had the ability to digest helper Ad-3I genome in vivo.
• Ad-3I helper virus was used for screening the selected cell clones.
• the digestion of Ad-3I genome which generates multiple small fragments in the Southern blot, is a indicator of the active I-Scel in the cell. Nearly all cell clones had the functional enzyme to digest the input adenovirus. On the other hand, in contrast to what observed in the in vitro digestion, the in vivo digestion seems not to be complete.
• the 2.8 kb fragment can be ready observed, which suggests that the second I-Scel site located between CMV promoter and ⁇ l -antitrypsin is not digested to 100%. This is probably due to the fact that cellular environment of mammalian cells is not the optimized condition as the in vitro digestion.
• Example 3 Production of Recombinant Adenovirus A. Generation of Helper-Dependent Vector
• Ad- ⁇ -lacZ A helper-dependent vector, Ad- ⁇ -lacZ, was produced essentially as described in Fisher et al, Virol, 217: 1 1-22 (1996).
• This vector contains the sequence from the left-end of Ad5 encompassing bp 1-360 (5' inverted terminal repeat (ITR), a lacZ minigene under the transcriptional control of the cytomegalovirus (CMV) immediate early enhancer/promoter, and sequence from the right end of Ad5 spanning bp 35,353 to the end of the genome (3' ITR).
• ITR inverted terminal repeat
• CMV cytomegalovirus
• This plasmid was designed so that digestion with EcoRI releases the terminal ends of the Ad- ⁇ -lacZ genome from the plasmid backbone.
• Helper virus Ad-3I was infected at MOI of 5. The cells were harvested at full cytopathic effect (CPE). After freezing and thawing the cells three times, the cell lysates were used for determination of virus titer by X-Gal staining, as described in Fisher et al, cited above. Cell lysates were also subjected to a second round of amplification to permit rescue of helper dependent vector Ad- ⁇ -lacZ, which was performed essentially as described by Fisher et al, cited above.
• FIG. 4 The performance of the 293 I-Scel cell line in production of a recombinant adenovirus deleted of all adenoviral genes is presented in Fig. 4.
• the deleted Ad vector, EcoRI-digested pAd- ⁇ -lacZ (10 ⁇ g) was transfected in 1 x 10 5 cells of each of three cell lines: a conventional 293 cell line and two independent clones of 293-I-SceI (termed (a) and (b)). After transfection, the cells were infected with Ad-3I helper virus at MOI 5. The cell lysates were harvested 96 hours after infection.
• a portion of the cell lysates were used to determine the titer of Ad- ⁇ -lacZ virus titer for the first round of amplification. The remainder of the cell lysate was used for a second round of amplification in the same cell lines.
• the titer is shown as lacZ forming unit (LFU) per field under microscopy under identical conditions. See Fig. 4.
• LFU lacZ forming unit
• the initial rescue of helper dependent viruses was better in 293 cells while subsequent amplification of helper virus is better in 293-I-SceI cell lines. This may reflect the importance in the initial rescue step of adequate amounts of helper virus. In the 293-I-SceI cell line, the amount of helper virus is decreased because of I-Scel digestion.
• helper virus In the amplification procedure, the balance of helper virus and vector is more critical.
• a further characterization of the Ad-3I-293-I-SceI system is shown in Fig. 5.
• the same amounts of helper virus and EcoRI-digested plasmid Ad- ⁇ -lacZ were used to infect the same amounts of 293 cells, 293-I-SceI(a) cells and 293-I-SceI(b) cells (two different clones).
• the amount of helper virus was decreased in the 293-I-SceI cell line.
• the helper virus yield is increased 10- 100 fold in a simple step of amplification.
• the plasmid pAAV-Scel was constructed by cloning the blunt ended EF-nuc-I-Scel cassette from pEF-Scel into pSub201 at the Xbal site. Infectious
• AAV-Scel was generated using the techniques described previously [Xiao, J. Virol, 72: 10222-10226 (1998)].
• 293 cells (1 x 10 5 ) are transfected with 10 ⁇ g of EcoRI-digested pAd- ⁇ -lacZ and rAAV-Scel (MOI 10). After transfection, the cells are infected with Ad- 31 at MOI 5. The cell lysates are harvested 96 hours after infection.
• the levels of reporter gene expression ( ⁇ l -antitrypsin gene expression) after Ad-3I infection (MOI from 0.1 , 1 , 10, 100, 200) in media from either 293 cells, the 293-I-SceI cell line, or 293 cells with rAAV-EF-I-Scel (MOI 10) co-infection were monitored at various time points by ELISA which was performed as described by [Xiao et al, J. Virol, 72: 10222-10226 (1998)].
• a Southern blot of the Hirt DNA was performed using a probe specific for human ⁇ l -antitrypsin cDNA. DNA fragments of 1.7 kb, 2.8 kb and 32 kb were obtained. These fragments are indicative of digestion with the I-Scel.
• the Hirt DNA was extracted 24 hours post infection and electrophoresed in 0.8% agarose gel. The results clearly showed that replication of helper virus, Ad-3I, was lowered dramatically in the presence of I-Scel gene expression in the cell line. Ad-3I genome is disabled by I-Scel restriction digestion, which most likely causes the decrease in the copy number of the ⁇ l -antitrypsin gene available for transcription. The Southern blot also further confirmed that this is the case.

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