Novel replication deficient adenovirus vectors and methods for making and using them
The invention provides novel replication deficient adenovirus vectors and methods for making and using these viruses. The invention also provides vector systems and kits using a serotype specific strategy for making adenoviral vector preparations substantially free of replication competent “helper” virus. The helper virus-free preparations provide novel pharmaceutical compositions substantially free of helper virus for use in gene transfer and gene therapy.
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[0001] This application is a continuation-in-part (CIP) of PCT Application Serial No. PCT/US01/02213, filed Jan. 22, 2001, which is a CIP of U.S. patent application Ser. No. (“U.S. Ser. No.”) 09/488,867, filed Jan. 21, 2000. The aforementioned applications are explicitly incorporated herein by reference in their entirety and for all purposes.
STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH FIELD OF THE INVENTION[0003] This invention generally pertains to the fields of virology, medicine and gene therapy. In particular, this invention provides novel replication deficient adenovirus vectors and vector systems and methods for making and using these viruses. The compositions and methods of the invention are used to make replication defective adenoviral gene therapy vector preparations that are substantially free of replication competent helper viruses.
BACKGROUND OF THE INVENTION[0004] Adenovirus vectors have become important tools in gene therapy and for the in vivo and ex vivo cell-targeted transfer of heterologous, therapeutic genes to diseased cells or tissues, including the treatment of genetic diseases and cancer. Several properties make adenovirus advantageous gene therapy vectors. They can be produced in high titer stocks. Adenovirus can infect resting and nondividing cells, such as dendritic cells and neurons. The adenoviral genome, which is a linear, double-stranded DNA, can be manipulated to accommodate foreign genes that range in size, including reasonably large DNA inserts. They can be re-targeted to a variety of cells. They do not require host cell proliferation to express adenoviral or transgene-encoded proteins. There are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses. As adenoviral vectors do not insert into the chromosome of a cell the effect is impermanent and less likely to interfere with the cell's normal function. Live adenovirus has been safely used for many years for human vaccines. Human adenoviruses have been used in humans as in vivo gene delivery vehicles.
[0005] However, available adenoviruses can also present serious problems when used in vivo. One drawback to adenovirus-mediated gene therapy is that decreases in gene expression are typically observed after about two weeks following administration of the vector. This loss of expression may require re-administration of the viral vector. If the same adenovirus serotype is re-administered, the host may generate neutralizing antibodies against the fiber or hexon proteins of the viral vector. Such a serotype specific anti-adenovirus response may prevent effective re-administration of the viral vector.
[0006] If viral replication is not desired, as with most gene therapy treatments, use of replication competent human adenoviruses is also problematic. For example, infection both in vivo and in vitro with the adenoviral vector can result in cytotoxicity to target cells due to the accumulation of penton protein, which is toxic to mammalian cells. Thus, in gene therapy, replication incompetent or replication defective (transgene-containing) adenovirus genomes are preferred over replication competent forms.
[0007] One approach to make a replication defective adenovirus is to inhibit viral DNA replication by disabling at least one necessary viral gene. Many currently available gene therapy adenovirus vectors are inactivated by deletion of the viral early gene region 1, (or “E1 gene”), E2A gene, E2B gene, or E4 gene. Complementation of the disabled gene by a second source will allow replication of the disabled virus. A commonly used method of complementation involves introducing the disabled (transgene-containing) adenoviral genomes into an adenoviral replication competent host cell that stably expresses the missing or mutated viral gene (e.g., the E1 gene). However, this approach has a significant risk in that the defect in the disabled genome can be replaced by homologous recombination with a wild type sequence to produce a replication competent variant.
[0008] To address problems created by homologous recombination with wild type sequences adenoviruses have been disabled by deleting most, if not virtually all, viral genes. Adenoviral vectors with only inverted terminal repeats flanking the genome (for DNA replication), an adenoviral packaging signal (to effect insertion of the completed viral genome into a completed viral capsid) and a heterologous transgene have been constructed. In this scheme, to replace the deleted viral genomic sequences a “helper” virus with the necessary complementary genes is expressed (co-transfected or co-infected) with the disabled virus in a host cell. However, this model of adenovirus disabling and complementation is inherently flawed because significant amounts of the “helper” (replication competent) adenovirus can be inadvertently encapsidated. Attempts to decrease the amount of “helper outgrowth” include attenuation of the packaging capability of the helper virus by full or partial deletion of the packaging signal in the helper virus. However, helper virus outgrowth still is a problem with these schemes.
[0009] Rapid advances in gene therapy have created a great demand for safe and effective adenoviral gene transfer vectors, particularly replication defective constructs. However, current methods for making replication defective adenoviral gene therapy vectors do not adequately address the problem of “helper outgrowth” contamination by replication competent virions. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION[0010] The present invention provides novel vectors and vector systems and methods for selectively packaging adenovirus nucleic acid sequences as replication defective virions based on adenovirus serotype. The serotype specific strategy prevents or actively blocks contamination of adenovirus replication defective vector preparations by replication competent helper viruses. The invention utilizes the 52/55 kDa protein and/or the IVa2 to modulate the production of contaminating replication-competent viral vectors.
[0011] The invention provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid based on adenovirus serotype, comprising: a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid; a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein; and a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein and lacking the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein.
[0012] In one embodiment, the first replication defective adenovirus sequence is a helper-dependent adenovirus sequence. In another embodiment, the second replication defective adenovirus sequence is a helper adenovirus sequence. In another embodiment a first adenovirus serotype cis-acting packaging sequence is a helper-dependent cis-acting packaging sequence. In yet another embodiment, a second adenovirus serotype cis-acting packaging sequence is a helper adenovirus cis-acting packaging sequence.
[0013] In this vector system, the adenovirus capsid, packaging and/or 52/55 kDa trans-acting protein encoding sequences can be human adenovirus sequences. Further, in this vector system, the adenovirus capsid, packaging and/or IVa2 trans-acting protein encoding sequences can be human adenovirus sequences. In this system, the first and second adenovirus serotypes can be adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), or adenovirus type 40 (Ad40), and the first serotype differs from the second serotype. In alternative embodiments, the first adenovirus serotype is adenovirus type 5 and the second adenovirus serotype is adenovirus type 7, or, the first adenovirus serotype is adenovirus type 7 and the second adenovirus serotype is adenovirus type 5. In one embodiment, the first replication defective adenovirus sequence is a helper-dependent adenovirus sequence. In another embodiment, the second replication defective adenovirus sequence is a helper adenovirus sequence. In one embodiment a first adenovirus serotype cis-acting packaging sequence is a helper-dependent cis-acting packaging sequence. In another embodiment, a second adenovirus serotype cis-acting packaging sequence is a helper adenovirus cis-acting packaging sequence.
[0014] In one embodiment, the first replication defective adenovirus sequence cannot produce a complete adenovirus capsid, e.g., because the defective adenovirus sequence lacks a nucleic acid needed to produce a complete adenovirus capsid. In alternative embodiments, the first replication defective adenovirus sequence is encapsidated in a capsid comprising at least one polypeptide encoded by the second replication defective adenovirus sequence, or, the first replication defective adenovirus sequence is encapsidated in a capsid encoded by the second replication defective adenovirus sequence. The replication defective adenovirus can comprise a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon polypeptide gene or a combination thereof.
[0015] In one embodiment of the vector system of the invention, the inability to produce a functional 52/55 kDa trans-acting protein or IVa2 trans-acting protein is due to a mutation in the sequence encoding the protein. The mutation can be a missense mutation, a point mutation, a frameshift mutation or a deletion mutation.
[0016] In another embodiment, the second replication defective adenovirus sequence can further comprise a nucleic acid sequence encoding a polypeptide having the activity of the first serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein.
[0017] The nucleic acid sequence encoding the polypeptide having the activity of the first serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein can further be associated with an adenovirus replication competent host cell. The adenovirus replication competent host cell can be, e.g. a 293 cell line.
[0018] In the vector system of the invention, the polypeptide having the activity of a first serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein can be a first serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein.
[0019] In one embodiment, the first replication defective adenovirus sequence can lack at least one nucleic acid sequence needed to produce a capsid and can further comprise a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or IVa2 trans-acting protein. In one embodiment a first replication defective adenovirus sequence is a helper-dependent adenovirus sequence.
[0020] The invention also provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid based on adenovirus serotype, comprising: a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid; and, a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein.
[0021] The invention also provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid based on adenovirus serotype, comprising: a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid; a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein; and, a cell comprising a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein.
[0022] The invention also provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid based on adenovirus serotype, comprising: a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid; a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein; and, an expression cassette comprising a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein.
[0023] The invention also provides a vector comprising a replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence, a nucleic acid sequence encoding a functional second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein, wherein the second trans-acting protein does not have the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein, lacking the ability to produce a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or first adenovirus serotype IVa2 trans-acting protein. In alternative embodiments, the vector can further comprise at least one adenoviral nucleic acid sequence needed to produce an adenoviral capsid, or, it can comprise sufficient adenoviral nucleic acid sequence to produce a complete adenoviral capsid when the vector is expressed in an adenovirus replication-competent host cell. The first and second adenovirus serotypes can be adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), or adenovirus type 40 (Ad40), and the first serotype differs from the second serotype. In alternative embodiments, the first adenovirus serotype is adenovirus type 5 and the second adenovirus serotype is adenovirus type 7, or, the first adenovirus serotype is adenovirus type 7 and the second adenovirus serotype is adenovirus type 5.
[0024] The invention also provides a transformed or isolated infected cell comprising the vector system or vector of the invention.
[0025] The invention also provides a kit for making adenovirus encapsidated replication defective sequences comprising: a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid, a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein, and, a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein.
[0026] In the kit, the nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein can further be associated with an adenovirus replication competent cell. The nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein can further comprise an expression cassette. The second replication defective adenovirus sequence can further comprise the nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein.
[0027] The invention also provides a method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: (a) transforming or infecting into adenovirus replication competent host cells (i) a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous gene, (ii) a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein, and, (iii) a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein; and, (b) culturing the cells under conditions where the first replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector. In one embodiment, the first replication defective adenovirus sequence is a helper-dependent adenovirus sequence. In another embodiment, the second replication defective adenovirus sequence is a helper adenovirus sequence. In one embodiment a first adenovirus serotype cis-acting packaging sequence is a helper-dependent cis-acting packaging sequence. In another embodiment, a second adenovirus serotype cis-acting packaging sequence is a helper adenovirus cis-acting packaging sequence.
[0028] The invention also provides a method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences, wherein the cell comprises a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein, (i) a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence and a heterologous gene, and, (ii) a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein; and, (b) culturing the cells under conditions where the first replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.
[0029] The invention also provides a method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences (i) a first replication defective adenovirus sequence comprising a first adenovirus serotype cis-acting packaging sequence, a heterologous gene and a nucleic acid sequence encoding a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein or a first adenovirus serotype IVa2 trans-acting protein, and, (ii) a second replication defective adenovirus sequence comprising a second adenovirus serotype cis-acting packaging sequence, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein or a second adenovirus serotype IVa2 trans-acting protein; and, (b) culturing the cells under conditions where the first replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.
[0030] In these methods of the invention, the second replication defective adenovirus sequence can further comprise sufficient adenoviral nucleic acid sequence to encode a complete adenoviral viral capsid.
[0031] The invention also provides a vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids based on adenovirus serotype, comprising a replication defective adenovirus sequence comprising an adenovirus serotype 5 (Ad5) cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 7 (Ad), adenovirus serotype 12 (Ad12) or adenovirus serotype 17 (Ad17) 52/55 kDa trans-acting protein, and sufficient adenoviral nucleic acid sequence to encode a viral capsid, lacking the ability to produce a polypeptide having the activity of an adenovirus 5 (Ad5) serotype 52/55 kDa trans-acting protein or an adenovirus 5 (Ad5) IVa2 trans-acting protein.
[0032] The invention also provides a vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids based on adenovirus serotype, comprising a replication defective adenovirus sequence comprising an adenovirus serotype 7 (Ad7) cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 5 (Ad5) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein, and sufficient adenoviral nucleic acid sequence to encode a viral capsid, lacking the ability to produce a polypeptide having the activity of an adenovirus 7 serotype (Ad7) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein.
[0033] The invention also provides a vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids based on adenovirus serotype, comprising a replication defective adenovirus sequence comprising an adenovirus serotype 12 (Ad12) cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 5 (Ad5) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein, and sufficient adenoviral nucleic acid sequence to encode a viral capsid, lacking the ability to produce a polypeptide having the activity of an adenovirus 12 serotype (Ad12) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein.
[0034] The invention also provides a vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids based on adenovirus serotype, comprising a replication defective adenovirus sequence comprising an adenovirus serotype 17 (Ad17) cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 5 (Ad5) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein, and sufficient adenoviral nucleic acid sequence to encode a viral capsid, lacking the ability to produce a polypeptide having the activity of an adenovirus 17 serotype (Ad17) 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein.
[0035] The invention also provides a pharmaceutical composition comprising an encapsidated replication defective adenovirus, made using the vector system of claim 1, substantially free of helper virus, and a pharmaceutically acceptable excipient. In alternative embodiments, the pharmaceutical composition is 100%, 99.99%, 99.98%, 99.97%, 99.95%, 99.93%, 99.9%, 99.5%, 99%, 98%, 97%, 95%, and 90% pure of helper virus.
[0036] The invention also provides a method of delivering a heterologous nucleic acid to a cell comprising transforming or infecting a cell with the pharmaceutical composition of the invention. The pharmaceutical composition can be administered to a patient systemically, regionally or locally.
[0037] The invention also provides a vector system for selectively packaging a replication defective nucleic acid sequence in a virus capsid including a helper-dependent adenovirus nucleic acid sequence that includes 5′ and 3′ viral inverted terminal repeats (ITRs); a first adenovirus serotype-specific cis-acting packaging sequence; and a heterologous nucleic acid wherein the helper-dependent adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence; a helper adenovirus nucleic acid sequence that includes 5′ and 3′ virus ITRs; a second adenovirus serotype-specific cis-acting packaging sequence, wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein specific for the second adenovirus serotype-specific cis-acting packaging sequence; and a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific 52/55 kDa trans-acting protein or an adenovirus IVa2 trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence.
[0038] The invention further provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, including a helper-dependent adenovirus nucleic acid sequence comprising 5′ and 3′ ITRs from a first adenovirus serotype; a cis-acting packaging sequence from a first adenovirus serotype; and a heterologous nucleic acid, wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype. The vector system further includes a chimeric helper adenovirus nucleic acid sequence comprising 5′ and 3′ adenovirus ITRs from a second adenovirus serotype; a cis-acting packaging sequence from a second adenovirus serotype; and an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype, wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype. In one embodiment, the first adenovirus serotype is Ad7. In another embodiment, the second adenovirus serotype is Ad5. The invention further provides a method of producing the replication defective encapsidated adenovirus gene transfer vector.
[0039] The invention further provides a chimeric helper adenovirus nucleic acid sequence including 5′ and 3′ adenovirus ITRs from a second adenovirus serotype; a cis-acting packaging sequence from a second adenovirus serotype; and an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype, wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype. In one embodiment, the second adenovirus serotype is Ad5. In another embodiment, the non-second adenovirus serotype is Ad7. In one embodiment the chimeric helper adenovirus nucleic acid sequence is designated Ad7/5ITR&PSgr;-GFP.
[0040] The invention further provides a cell line for packaging a chimeric helper adenovirus nucleic acid sequence including a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype. In one embodiment, the second adenovirus serotype is Ad5. In another embodiment, the cell line is a 293 cell line. In yet another embodiment, the cell line is designated 293-L1.
[0041] The invention further provides a vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid including a helper-dependent adenovirus nucleic acid sequence comprising 5′ and 3′ ITRs from adenovirus serotype Ad7; a cis-acting packaging sequence from adenovirus serotype Ad7; and a heterologous nucleic acid, wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad7. The vector system further includes a chimeric helper adenovirus nucleic acid sequence comprising 5′ and 3′ adenovirus ITRs from adenovirus serotype Ad5; a cis-acting packaging sequence from adenovirus serotype Ad5; and an adenovirus serotype-specific nucleic acid sequence from adenovirus serotype AD7, wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad5.
[0042] A further understanding of the nature and advantages of the present invention is realized by reference to the remaining portions of the specification, the figures and claims.
[0043] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS[0044] FIG. 1 shows the sequence of the N-terminus of the Ad5 52/55 kilodalton protein open reading frame (ORF) in upper case letters; directly below is the corresponding amino acid sequence. Below that in lower case are the point mutations that introduce a series of stop codons, indicated by asterisks.
[0045] FIG. 2 is a reproduction of a radiograph showing the results of a Southern blotting experiments, as explained in detail in Example 2, below. Human 293 cells, or, in lane 3, 293-L1 cells, which express the Ad5 L1 52/55 kDa protein, were infected with the virus(es) indicated at the top of each lane (“5” represents an Ad5 serotype, “12” represents an Ad12 serotype, “17” represents an Ad17 serotype). The arrows point to the two bands that are diagnostic of packaging of the pm8001 (&Dgr;L1) adenoviral DNA.
[0046] FIG. 3 shows that other serotypes cannot complement the packaging defect of pm800. (A) Restriction maps of Ad5 and pm8001. KpnI and SpeI sites are indicated by lines and arrowheads, respectively. (B), (C) 293 or 293-L1 (Lane 2 in both B and C) cells were infected with the indicated viruses at an MOI of 5 pfu/cell. Forty eight hours post-infection, DNAs were extracted from purified virions and digested with KpnI and SpeI. Southern blots were performed to determine the serotype of the DNAs in the virions. In panel B, the arrow points to the Ad5-specific band containing the L1 gene, which is digested by SpeI in pm8001 DNA to yield the two bands indicated by the arrowheads.
[0047] FIG. 4 shows that pm8001 replicates its DNA and assembles capsids in cells co-infected with Ad7. (A) 293 cells were co-infected with the indicated viruses at an MOI of 5 pfu/cell. At the indicated times post-infection (hpi), low molecular weight DNAs were extracted from the cells and digested with KpnI and SpeI. Southern blots were performed to determine the amount of viral DNA in the infected cells. The ratio of the signals in bands A and B is shown below each lane. (B) Empty capsids or mature virions were isolated from infected cells. 5×1010 particles were boiled in sample buffer and loaded on a SDS-PAGE gel. Immunoblotting was performed to detect pm8001 empty capsids using an Ad5 IVa2 protein-specific monoclonal antibody. Lane 1, empty capsids from pm8001 infected cells; lane 2, empty capsids from pm8001 and Ad7 co-infected cells; lane 3, mature virions from Ad7 infected cells; lane 4, 1 &mgr;g of Ad7-infected whole cell lysate; lane 5, 1 &mgr;g of Ad5-infected whole cell lysate.
[0048] FIG. 5 shows the genomic structure of Ad7/5ITR&PSgr;-GFP.
[0049] FIG. 6 shows the Ad7/5ITR&PSgr;-GFP virus replicates its DNA in 293 cells. 293 cells were transfected with 5 &mgr;g PmeI-digested pW120700. Viral DNAs were isolated at 1d, 3d, and 7d post-transfection (lane 2-4, respectively) and digested with BclI. Southern blotting was performed to detect viral DNA. Lane 1, BclI-digested pW120700. Lane 5, BclI-digested wild type Ad7 DNA.
[0050] FIG. 7 shows the Ad5 52/55-kDa protein does not support the growth of Ad7/5ITR&PSgr;-GFP. 293 (A, B) or 293-L1 (C, D) cells were transfected with 10 &mgr;g PmeI-digested pW120700. Expression of GFP was examined using a fluorescent microscope at 2 d (A, C) and 11 d (B, D) post-transfection. Magnification=200×
[0051] FIG. 8 shows that the Ad5 IVa2 protein supports the growth of Ad7/5ITR&PSgr;-GFP. 293 (A-E) or 293 L1 (F-J) cells were co-transfected with 10 &mgr;g pW120700 and 10 &mgr;g pBK-TripIVa2. GFP expression was examined using a fluorescent microscope at 2 d (A), 3 d (F), 11 d (B, C, G, H), and 14 d (D, E, I, J) post-transfection. Panels C, E, H, and J are the same fields as B, D, G, and I, respectively, taken using both fluorescent light and regular light.
[0052] FIG. 9 shows that Ad5 IVa2-expressing 293 cells produce infectious Ad7/5ITR&PSgr;-GFP virus. Viral lysates made from 293 cells 14 days after co-transfection with PmeI-digested pW120700 and pBK-TripIVa2, were used to infect fresh 293 (A, C, E) or 293-IVa2 (B, D, F) cells. Expression of GFP was examined using a fluorescent microscope at 3 days (A, B) and 6 days (C, D) post-infection. Panels E and F are of the same fields as C and D, respectively, taken using both fluorescent light and regular light.
DETAILED DESCRIPTION[0053] The present invention provides a novel strategy to produce preparations of safe and effective replication defective adenovirus (Ad) that are substantially free of replication competent forms. Such “helper virus”-free preparations are particularly useful as gene transfer vectors and gene therapy vectors for administration as pharmaceuticals. The invention is based on the discovery that the mechanism by which the adenovirus packages its newly replicated genome into a newly formed capsid is dependent on serotype specific cis- and trans-acting packaging factors.
[0054] A first step in adenovirus replication is the separate synthesis of a complete viral genome and a complete viral capsid. The completed genome is then inserted, or “packaged,” into the finished capsid. The exact mechanism used by the virus to package a completed genome into a completed capsid to make a mature virion is not known. However, it is known that packaging of the adenovirus genome requires at least two elements.
[0055] First, there is a specific set of sequences, or motifs, mapping at the left end of the genome, which serve as the viral packaging signal. Adenoviral genomes without a packaging signal will not be inserted into a capsid structure. This packaging signal region can contain at least five functionally redundant domains, called “A repeats” (see, e.g., Grable (1990) J. Virol. 64:2047-2056; Grable (1992) J. Virol. 66:723-731). “A repeats” contain consensus motifs that may be able to function independently (Schmid (1997) J. of Virol. 71:3375-3384; Schmid (1998) J. Virol. 71:3375-3384). These motifs appear to be conserved among different adenovirus serotypes.
[0056] Second, a soluble trans-acting protein factor of about 52 to 55 kilodaltons (kDa), called the “52/55 kDa protein,” must be present during the replication process to form mature virions, i.e., capsids containing complete adenoviral genomes. Exactly how the 52/55 kDa protein is involved in the genome encapsulation process is not known. A trans-acting protein may bind directly, alone or with another protein, to the packaging signal. However, if a functional 52/55 kDa protein is not present, the completed adenoviral genome will not be inserted into a finished capsid structure (see, e.g., Gustin (1998) J. Virol. 72:7860-7870). The 52/55 kDa proteins show significant homology from serotype to serotype over most of the molecule; however, there are less well-conserved sequences at the amino and carboxyl termini (K. Gustin, Ph.D. Thesis, University of Michigan, 1998). These non-conserved amino and carboxyl termini may be involved in the serotype-specific binding of 52/55 kDa protein to cis-acting sequences or other trans-acting factors.
[0057] As published findings have reported that “A repeat” motifs appear conserved among different adenovirus serotypes and that the 52/55 kDa protein shows fairly limited homology from serotype to serotype, it was a surprising discovery that the adenoviral packaging cis- and trans-elements act in a serotype specific manner. For example, adenoviral type 7 (Ad7) serotype 52/55 kDa protein will not support genome encapsidation in combination with an adenoviral type 5 (Ad5) serotype packaging signal. The invention for the first time combines a replication defective gene therapy vector having a first cis-acting packaging signal with a helper virus having a second cis-acting packaging signal, wherein the gene therapy vector encodes a 52/55 kDa trans-acting protein only able to support the packaging activity of a first cis-acting packaging signal (leaving the helper virus without a functional encapsidation apparatus).
[0058] Typically, the 52/55 kDa trans-acting protein only able to support the packaging activity of the second cis-acting packaging signal is a 52/55 kDa polypeptide derived from the same serotype as that of the second cis-acting signal. This novel strategy provides a means of making replication defective virion preparations substantially free of replication competent “helper” virus (i.e., little to no “helper outgrowth” contamination). Thus, the invention provides novel adenoviral vectors and vectors systems for producing replication deficient vectors. It also provides novel means of making and using these vectors.
[0059] In the experiments described in Example 1, below, an Ad5 serotype virus lacking a 52/55 kDa polypeptide-encoding region (“pm8001”) could be complemented in trans using 52/55 kDa proteins from a matching Ad5 serotype; however, a non-matching Ad7 serotype could not complement the defect. Adding a serotype-matched Ad5 52/55 kDa protein with an Ad5 cis-acting packaging sequence allowed packaging of Ad5 nucleic acid; however, 52/55 kDa protein from a different serotype (Ad7) could not complement the pm8001 mutation. Experiments set forth in Example 2, below, further demonstrated this point: adding serotype mis-matched Ad12 or Ad 17 52/55 kDa protein in trans with pm8001 (Ad5 serotype lacking a 52/55 kDa protein) under replication permissive conditions did not allow packaging of nucleic acid comprising Ad5 (pm8001) packaging sequence (i.e., could not complement the pm8001 mutation).
[0060] In addition, it has been demonstrated that the Ad5 52/55-kDa protein is required for viral DNA encapsidation and interacts with the viral IVa2 protein (Gustin and Imperiale, (1998) J.Virol. 72:7860-7870). The IVa2 protein binds to critical DNA motifs in the packaging sequence (Zhang (2000) J.Virol. 74:2687-2693), indicating that the IVa2 protein is involved in viral DNA encapsidation. The data set forth in Examples 3-6 further indicate that the IVa2 is involved in the adenovirus packaging process.
[0061] The inventors have constructed a chimeric virus (Ad7/5ITR&PSgr;-GFP) containing an Ad7 genome except for the ITRs and packaging sequence, which were derived from Ad5. This virus can replicate its DNA and express its genes in 293 cells, but no infectious viruses are produced. 293 cells expressing the Ad5 L1 52/55 kDa protein cannot support the growth of the virus, while 293 cells expressing the Ad5 IVa2 protein can, indicating that the IVa2 protein plays a role in viral DNA packaging, and that a functional interaction between the IVa2 protein and the adenovirus packaging machinery is serotype-specific.
[0062] Co-infection data presented below further demonstrate that the Ad5 52/55-kDa protein is not required for the growth of the chimeric virus, since the Ad5 IVa2 protein alone in 293 cells supports its growth. These results indicate that the Ad7 packaging system, including its 52/55-kDa protein, can use the Ad5 IVa2 protein to package DNAs that contain the Ad5 packaging sequence. Moreover, the chimeric virus replicates its DNA in 293 cells, indicating that the Ad5 E1 proteins expressed in 293 cells activate Ad7 early gene expression, and that the Ad7 replication machinery can replicate DNA using the Ad5 ITRs. The human adenovirus ITRs are about 100 to 200 bp long. However, full replication requires only the first 45-70 base pairs of the ITR. The ITR contains a 10 base pair functional domain that is identical in all human adenovirus ITRs (Tamanoi (1983) Proc. Natl. Acad. Sci.USA. 80:6446-6450.). It has been shown that Ad2-infected cell extracts can replicate viral DNA from subgroups A-E in vitro, and that Ad4-infected cell extracts can replicate DNA containing the Ad2 ITR in vitro.
[0063] Ad7 and Ad5 are members of adenovirus subtypes B and C, respectively, whose sequences diverge significantly. The packaging sequences of Ad7 and Ad5 are 68% identical overall although the motifs previously demonstrated to be important for IVa2 binding in Ad5 are present in the Ad7 DNA packaging domains. In addition, the two 52/55-kDa proteins show only 70% identity and the IVa2 proteins 83%. Similar levels of sequence identity exist between Ad5 and Ad12 or Ad17.
[0064] The demonstration of serotype specific viral DNA packaging has important implications for the development of improved adenovirus gene transfer vectors. Gutted or helper-dependent adenovirus vectors have demonstrated great promise for prolonged therapeutic gene expression and reduced host immune responses. Currently, the utility of these vectors is limited by the fact that their growth requires a helper virus, and contamination by the helper virus cannot be totally eliminated by physical separation techniques or other manipulations. To date, investigators have either made the gutted chromosome smaller than that of the helper virus chromosome, resulting in slight differences in density that can be resolved somewhat in CsCl gradient, used a mutant packaging signal on the helper virus chromosome that reduces its ability to be packaged, or introduced loxP sites on either side of the packaging sequence of the helper virus and grown the vector in cells that express Cre recombinase. Such approaches still leave significant levels of helper virus contamination, however. The present invention provides methods, vectors and systems in which vector DNA (i.e., helper dependent nucleic acid) is specifically packaged without the packaging of the helper virus. For example, the chimeric virus can be used as a helper virus, which can be grown in 293 cells, or any other suitable cell line, expressing the Ad5 IVa2 protein. The helper-dependent vector can be derived from, for example, Ad7. When co-infecting both the helper virus and the helper-dependent vector in 293 cells only the vector DNA should be packaged providing the IVa2 protein present in the cell is specific for the cis-acting packaging sequences present in the helper-dependent vector.
[0065] Definitions
[0066] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0067] The term “adenovirus” or “Ad” includes all adenoviruses, including all members of the known six subgenera, classified as A to F, and the 47 known distinct human serotypes, as described in detail below. The term “adenovirus serotype” means the individual members of this viral genus that are defined and identified by their expression of at least one serotype-specific epitope. The invention incorporates the different cis- and trans-acting Ad genome packaging factors from all Ad serotypes, including all human and non-human strains.
[0068] The term “adenovirus serotype cis-acting packaging sequence” means the set of sequences mapping at the left end of the Ad genome which serve as cis-acting, serotype-specific packaging signals for inserting the Ad genome (or any nucleic acid comprising the packaging signal) into a complete Ad viral capsid, as described in detail below. The term includes the cis-acting packaging signal sequences of any size or variation that retain their packaging and serotype specific characteristics. The term includes the cis-acting packaging signal sequences from any serotype, including those from strains whose packaging factors have not been characterized in detail; these cis- (and trans-) acting packing factors can be identified either by sequence identity (homology) to known cis-acting packaging signal nucleic acid from other serotypes and/or by functional assays, as described in detail below.
[0069] The term “adenovirus serotype 52/55 kDa trans-acting protein” or “52/55 kDa protein” means a soluble trans-acting protein factor that must be present during the Ad replication process to complement the above-described cis-acting packaging signal to encapsidate the Ad genome in a serotype specific manner. The term includes polypeptides that are actually 52 to 55 kDa in size and functional (serotype specificity and packaging) equivalents, which can include smaller fragments of the wild type polypeptide. The term includes 52/55 kDa protein-encoding sequences from any serotype, including those from strains whose packaging factors have not been characterized in detail; these can be identified either by sequence identity (homology) to previously characterized 52/55 proteins from other serotypes and/or by functional assays, as described in detail below.
[0070] The phrase “polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein” includes any polypeptide that has the trans-acting packaging activity (i.e., ability to support encapsidation with the cis-acting sequence) of a second adenovirus serotype 52/55 kDa trans-acting protein but not (to a significant degree) the packaging activity of a first adenovirus serotype 52/55 kDa trans-acting protein. The invention is based on the surprising finding that a cis-acting packaging signal does not act in conjunction with a trans-acting 52/55 kDa polypeptide from any serotype; it will only package Ad genomes when matched with a 52/55 kDa polypeptide from the same (or a limited set of) serotypes. For example, an Ad5 cis-acting packaging sequence will be complemented by an Ad5 a trans-acting 52/55 kDa polypeptide but not (to a significant degree) by an Ad7 a trans-acting 52/55 kDa polypeptide. Thus, one exemplary vector system of the invention includes a gene therapy vector with an Ad5 cis-acting packaging signal and a helper virus with an Ad7 cis-acting packaging signal, with the system complemented by only an Ad5 a trans-acting 52/55 kDa polypeptide. However, a trans-acting 52/55 kDa polypeptide from another serotype may also be able to significantly complement the Ad5 cis-acting sequence. It can be used in that capacity in the compositions and methods of the invention as long as it cannot also complement (to a significant degree) the cis-acting sequence in the helper virus (the Ad7 cis-acting sequence in this example). Determining if a cis-acting sequence from one serotype can or cannot be functionally complemented by a trans-acting factor from another serotype can be determined by routine screening of cis- and trans-acting packaging factors (and variations thereof) derived from different serotypes using methods described herein.
[0071] The term “heterologous” when used with reference to a nucleic acid, indicates that the nucleic acid is in a cell or a virus where it is not normally found in nature; or, comprises two or more subsequences which are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged in a manner not found in nature; e.g., a human gene as a transgene operatively linked to a promoter sequence inserted into an adenovirus-based vector of the invention. As another example, the transgene can be a cytotoxin, wherein the adenovirus is administered for the treatment of cancer. Heterologous sequences can comprise various combinations of promoters and sequences (e.g., transgenes), examples of which are described in detail herein.
[0072] The term “inverted terminal repeat sequence” or “ITR” refers to the common usage of the term with respect to adenoviruses and includes all ITR sequences and variations thereof that are functionally equivalent, i.e., the term refers to sets of sequences (motifs) which flank (on the right and left) the linear adenovirus genome and are necessary for replication of the adenovirus genomic nucleic acid. The Ad sequences of the vectors and vector systems of the invention are flanked by ITRs. There is a high degree of sequence conservation within the ITR between adenoviruses of different serotypes, see, e.g., Schmid (1995) Current Topic in Microbiol. and Immunol. 199(Pt.1):67-80.
[0073] The phrase “lacking the ability to produce a functional adenovirus serotype 52/55 kDa trans-acting protein” means that no 52/55 kDa protein is produced or the 52/55 kDa protein that is produced cannot support the encapsidation of a nucleic acid comprising a cis-acting packaging signal of the same Ad serotype into a complete Ad capsid.
[0074] The term “nucleic acid” or “nucleic acid sequence” refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol 600, Eds. Baserga et al. (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press), WO 97/0321 1; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156.
[0075] The phrase “nucleic acid sequence encoding” refers to a nucleic acid comprising sequence that encodes a protein or peptide. It can also include translational or transcriptional regulatory element sequence. The nucleic acid sequence can include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences can include degenerate codons of a native sequence or sequences that may be introduced to provide codon preference in a specific host cell.
[0076] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. The pharmaceutical compositions of this invention are formulations that comprise a pharmacologically effective amount of a composition comprising a vector or combination of vectors of the invention (i.e., a vector system) and a pharmaceutically acceptable carrier.
[0077] As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant means” also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of polypeptide coding sequences in the vectors of invention.
[0078] The term “replication deficient” or “replication defective” refers to a viral genome that does not comprise all the genetic information necessary for replication and formation of a genome-containing capsid in a replication competent cell under physiologic (e.g., in vivo) conditions.
[0079] The invention provides a pharmaceutical composition comprising an encapsidated replication defective adenovirus substantially free of helper virus. The term “substantially free of helper virus” or “substantially free of replication competent virus” means that less than about 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or about 1.0% of the capsids in a preparation (e.g., the product of an infection by the vector system of the invention) can replicate in a replication competent cell without some form of complementation by another source, such as the cell, another virus, a plasmid, and the like. In alternative embodiments, pharmaceutical compositions are 100% pure, and about 99.99%, 99.98%, 99.97%, 99.96%, 99.95%, 99.93%, 99.90%, 99.5%, 99.0%, 98%, 97%, 95%, and 90% pure of helper virus.
[0080] The term “replication competent cell” or “replication competent host cell” or “producer cell” includes any cell capable of supporting the replication of an adenoviral genome and capsid and the encapsidation process. Typically, these cell lines are anchorage dependent viral packaging cell lines. Anchorage dependent cells, or cultures derived from them, are those that will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. Anchorage dependent cell lines commonly used for mammalian cell culture can be used as viral packaging cell lines. Examples of such anchorage dependent cell lines are HeLa cells; 911 cells (see, e.g., Fallaux (1996) Hum. Gene Ther. 7:215-222); 293 cells (see, e.g., Graham (1977) J. Gen. Virol. 36:59-72) and PER.C6 cells (see, e.g., WO/97/00326).
[0081] The term “sufficient adenoviral nucleic acid sequence to encode a viral capsid” means an adenoviral genome comprises sufficient genetic information to form a complete viral capsid in a replication competent cell under physiologic (e.g., in vivo) conditions.
[0082] The term “adenovirus (Ad) serotype IVa2 trans-acting protein” or “IVa2 protein” means a soluble trans-acting protein factor that supports the Ad replication process to complement the above-described cis-acting packaging signal to encapsidate the Ad genome in a serotype specific manner. The term includes polypeptides that are functional (serotype specificity and packaging) equivalents, which can include smaller fragments of the wild type polypeptide. The term includes IVa2 protein-encoding sequences from any serotype, including those from strains whose packaging factors have not been characterized in detail; these can be identified either by sequence identity (homology) to previously characterized IVa2 proteins from other serotypes and/or by functional assays, as described in detail below. In one aspect of the compositions and methods of the invention, the IVa2 protein can act alone to effect serotype specific Ad encapsidation (packaging). In alternative aspects, the IVa2 protein acts in conjunction with the adenovirus serotype 52/55 kDa trans-acting protein, discussed above, to effect serotype specific Ad encapsidation (packaging).
[0083] The invention provides pharmaceutical compositions for in vitro and for in vivo systemic, regional and local administration. The term “systemic administration” refers to administration of a composition of the invention, such as the Ad vectors and vector systems of the invention, in a manner that results in the introduction of the composition(s) into the circulatory system. The term “regional administration” refers to administration of a composition into a specific anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ, and the like. For example, regional administration includes administration of the composition into the hepatic artery for regional administration to the liver. The term “local administration” refers to administration of a composition into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous injections, intramuscular injections, and the like. Any one of skill in the art would understand that local administration or regional administration also can result in entry of the composition into the circulatory system.
[0084] Adenovirus DNA, Genomes and Virions
[0085] This invention provides novel engineered adenovirus genomes for use in the production of preparations of replication defective Ad gene transfer vectors that are substantially free of replication competent virus, such as helper virus. As the genes and vectors of the invention can be made and expressed in vitro or in vivo, the invention provides for a variety of means of making and expressing these genes and vectors. One of skill will recognize that desired phenotypes associated with altered gene activity can be obtained by modulating the expression or activity of the genes and nucleic acids (e.g., ITRs, promoters) within the vectors of the invention. Any of the known methods described for increasing or decreasing expression or activity can be used for this invention. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature.
[0086] General Techniques
[0087] The nucleic acid sequences of the invention and other nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to mammalian cells, e.g., bacterial, yeast, insect or plant systems.
[0088] Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
[0089] Techniques for the manipulation of nucleic acids, such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0090] Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g. fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
[0091] Structure and Properties of Adenovirus
[0092] Capsid Structure
[0093] In the vector system of the invention the first replication defective adenovirus sequence comprising a first Ad serotype cis-acting packaging sequence and a heterologous nucleic acid, flanked by ITRs, can lack some or all of the genetic information necessary to synthesize a capsid structure (described below). The first replication defective adenovirus sequence can be a helper-dependent adenovirus sequence. A first adenovirus serotype cis-acting packaging sequence can be a helper-dependent cis-acting packaging sequence. The deleted sequences can be completely or partially complemented by the second replication defective adenovirus, an Ad replication-competent cell, a third nucleic acid (e.g., a plasmid or virus), or a combination thereof. In one vector system of the invention, the first replication defective adenovirus sequence is encapsidated in a capsid comprising polypeptides encoded by the second replication defective adenovirus sequence (the “helper virus”) when both sequences are expressed in an adenovirus replication-competent host cell.
[0094] All adenovirus virions are nonenveloped icosahedral capsids surrounding a linear double stranded DNA genome. Virions are about 80 to 110 nm in diameter with about 252 capsomers each about 8 to 10 nm in diameter. The 240 nonvertex capsomers (hexons) are formed by the interaction of three identical polypeptides. The atomic structure of hexon polypeptide as determined by X-ray crystallography (Roberts (1986) Science 232:1148-1151) indicates that it has two distinct parts: a triangular top with three towers exposed to the environment and bearing type-specific antigenic determinants (Toogood (1992) J. Gen. Virol. 73:1429-1435) and a pseudo hexagonal base with a central cavity. Penton polypeptides form the 12 vertices of the viral icosahedron, a pentamer of penton bases tightly associated with a trimer of fibres projecting from the surface.
[0095] Adenovirus Serotypes
[0096] The invention provides adenovirus vectors, Ad genes and Ad subsequences derived from all Ad serotypes. The vectors of the invention include genomes and nucleic acid sequences from all of the family Adenoviridae including avian, human and other mammalian strains and serotypes. The family Adenoviridae is currently divided into two genera named Mastadenovirus and Aviadenovirus. All adenoviruses are morphologically and structurally similar. The human adenoviruses (Ads) belong to the genus Mastadenovirus. Human adenoviruses show diverging immunological properties and are divided into serotypes and, to date, 47 distinct serotypes have been identified. These are divided into six subgenera (A to F) on the basis of shared immunological and biochemical properties. Different serotypes are associated with a variety of acute infections, primarily respiratory, ocular, and gastrointestinal. For example, serotypes Ad 40 and Ad 41 can be isolated in high yield from feces of young children with acute gastroenteritis. The invention also includes use of newly isolated serotypes that can be identified using routine screening (see, e.g., Itoh (1999) J. Med. Virol. 59:73-77) or human cell-infecting non-human adenovirus (see, e.g., Rasmussen (1999) Hum Gene Ther. 10:2587-2599, describing canine Ad 2 and bovine Ad 3 infecting human cells).
[0097] Many adenovirus serotype genomes have been isolated and characterized (see, e.g., Chroboczek (1992) Virology 186: 280-285, comparing the Ad5 and the Ad2 genomes). For example, in Ad2 the genome is 35,937 base pairs long; see, e.g., Akusjarvi, G. et al. (1986) Structure and function of the adenovirus genome, In Adenovirus DNA, Doerfler, W., Ed., Martinus Nijhoff Publishing, Boston. The complete Ad serotype 17 (Ad 17) genome is described by Chillon (1999) J. Virol. 73 (3), 2537-2540, GenBank accession no. AF108105; for the Ad 40 complete genome see GenBank accession no. L19443; for the Ad 12 complete genome see GenBank accession no. X73487, and the like. See also, e.g., National Library of Medicine, and, Index Virum at http://life.anu.edu.au/viruses/Ictv/fr-index.htm. For non-human adenoviruses, see, e.g., Reddy (1999) J. Gen. Virol. 80:563-70; Matiz (1999) Virus Res. 55:29-35.
[0098] Genetic Engineering of Novel Adenovirus Vectors
[0099] The invention provides novel vector systems for serotype-specific, selective packaging (encapsidation) of adenovirus nucleic acid sequences in replication defective virions. The vector systems of the invention produce Ad gene therapy vector preparations that are substantially free of replication competent “helper virus.” The vector system comprises two replication defective adenovirus constructs. One of the replication defective adenovirus constructs comprises a first adenovirus serotype cis-acting packaging sequence and a heterologous nucleic acid, flanked by adenovirus inverted terminal repeat (ITR) sequences, lacking the ability to produce a polypeptide having the activity of a first adenovirus serotype 52/55 kDa trans-acting protein. In one embodiment, Ad sequences have been deleted from the Ad genome used to make this vector. In another embodiment, all Ad sequences except for the flanking ITRs and the Ad serotype-specific cis-acting packaging sequence have been deleted.
[0100] The second construct comprises a second adenovirus serotype cis-acting packaging sequence, flanked by adenovirus ITR sequences, lacking the ability to produce a polypeptide having the activity of a second adenovirus serotype 52/55 kDa trans-acting protein. In one embodiment, the second “helper virus” construct comprises some or all of the genomic sequence to allow complete capsid synthesis, assembly and packaging (in conjunction with the serotype-matched packaging factors).
[0101] The invention also provides a vector for selectively packaging Ad nucleic acid sequences in replication defective virions comprising a replication defective Ad sequence comprising a first Ad serotype cis-acting packaging sequence, a nucleic acid sequence encoding a polypeptide having the activity of a second Ad serotype 52/55 kDa trans-acting protein, and sufficient Ad nucleic acid sequence to encode a viral capsid, flanked by inverted terminal repeat sequences.
[0102] To allow for packaging of the heterologous nucleic acid (transgene)-containing construct (with a first serotype specific cis-acting sequence), the vector system includes a nucleic acid sequence encoding a 52/55 kDa trans-acting protein that can functionally complement the first cis-acting packaging signal (to allow for encapsidation). This polypeptide can be inserted into the second “helper virus” construct or it can be expressed in the Ad replication competent cell. In another embodiment, this 52/55 kDa polypeptide-encoding sequence is inserted in the transgene-containing construct having the first cis-acting packaging sequence. In this latter example the packaged virion must be replication defective; thus, it must also lack at least one functional gene or polypeptide needed for replication in a replication competent cell. In one embodiment, all virion genes are deleted or disabled except for the flanking ITRs and the cis-acting packaging signal.
[0103] While adenovirus vectors are normally trophic for the respiratory epithelium (Straus, In Adenoviruses, Plenan Press, New York, pp. 451-496 (1984), the capsid proteins can be designed to re-target the gene therapy virus to a variety of different cell types. Chimeric Ad coat proteins, e.g., a fiber, hexon or penton protein (see above), can be so engineered by introduction of a nonnative amino acid sequence, e.g., at or near the carboxyl terminus. Re-targeting is accomplished by, e.g., recombinantly engineering a heterologous cell targeting ligand into the Ad fiber, hexon or penton receptor gene; see, e.g., Gonzalez (1999) Gene Ther. 6:314-20; U.S. Pat. No. 5,965,541. Examples of such modifications are described in Wickham (1997) J. Virol 71:8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Arnberg. (1997) Virology 227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris (1996) TIG 12:400-405; Stevenson (1997) J. Virol. 71:4782-4790; Michael (1995) Gene Therapy 2:660-668 (incorporation of gastrin releasing peptide fragment adenovirus fiber protein); and Ohno (1997) Nature Biotechnol. 15:763-767 (incorporation of Protein A-IgG binding domain into Sindbis virus). Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g. Michael (1993) J. Biol. Chem. 268:6866-6869; Watkins (1997) Gene Therapy 4:1004-1012; Douglas (1996) Nature Biotechnol. 14: 1574-1578. Any protein or other composition can be conjugated to the viral surface to achieve targeting, see, e.g. Nilson (1996) Gene Therapy 3:280-286 (conjugation of EGF to retroviral proteins).
[0104] Thus, gene therapy using the pharmaceuticals of the invention can be carried out in the treatment of diseases, disorders, or conditions associated with different tissues that normally lack receptors to which wild-type Ad binds, e.g., for delivery to monocyte/macrophages, fibroblasts, neuronal, smooth muscle, and epithelial cells. Such tissues (and associated diseases, disorders, or conditions) include, e.g., endothelia (e.g., angiogenesis, restenosis, inflammation, and tumors); neuronal tissue (e.g., tumors, chronic pain, Alzheimer's disease); epithelium (e.g., disorders of the skin, cornea, intestine, lung); hematopoietic cells (e.g., human immunodeficiency virus (HIV-1, HIV-2), cancers, anemias); smooth muscle (e.g., restenosis, degeneration, heart disease); fibroblasts (e.g., inflammation, scarring, delayed healing).
[0105] Methods to generate and replicate these hybrid constructs are well known in the scientific and patent literature, see, e.g., U.S. Pat. No. 5,981,225; U.S. Pat. No. 5,922,576; U.S. Pat. No. 5,880,102; Graham (1995) Mol. Biotechnol. 3:207-220, and for general methodologies, e.g., Sambrook, Ausubel, Tijssen. Viral genome genetic engineering, transformation and infection techniques in cell culture, viral manipulation and isolation techniques, Ad replication competent cell lines and permissive conditions for Ad replication, and the like, are all well known and described in the scientific and patent literature, see e g., Krougliak (1995) Hum. Gene Ther. 6:1575-1586; Gorziglia (1999) J. Virol. 73:6048-6055; Cote (1998) Biotechnol. Bioeng. 59:567-575; Hartigan-O'Connor (1999) J. Virol. 73:7835-7841; U.S. Pat. No. 5,851,806; U.S. Pat. No. 5,880,102; U.S. Pat. No. 5,882,877; U.S. Pat. No. 5,891,690; U.S. Pat. No. 5,965,541; U.S. Pat. No. 5,981,225; U.S. Pat. No. 5,985,846; U.S. Pat. No. 5,994,106; U.S. Pat. No. 5,955,281.
[0106] To make a null mutant in a structural protein (e.g., the 52/55 kDa protein) or a structural motif (e.g., ITR or packaging sequence) base substitutions can be inserted in the open reading frame (ORF), e.g., near the 5′ end to create a series of stop codons. Alternatively, complete deletion of genomic sequence can be made (e.g., deletion of genes involved in capsid synthesis, as hexon or penton coding sequences). The engineered ORFs are typically manipulated using both prokaryotic and eukaryotic expression vectors. Manipulated (e.g., mutated or partially deleted) sequences are then inserted back onto an adenovirus chromosome.
[0107] Recombinantly engineered adenoviral vectors can be generated by a variety of known procedures, e.g., in vivo homologous recombination method (see, e.g., He (1999) Proc. Natl. Acad. Sci. USA 95:2509-2514; Aoki (1999) Mol. Med. 5:224-231; Souza (1999) Biotechniques 26:502-508; U.S. Pat. No. 5,919,676), by the in vitro direct ligation method (see, e.g., Mizuguchi (1998) Hum. Gene Ther. 9:2577-2583, or using circular adenoviral DNA (see, e.g., Tashiro (1999) Hum. Gene Ther. 10:1845-1852). One exemplary technique, the altered sequences are inserted in a bacterial clone taking advantage of a bacterial recombination system, e.g., as the method described by Chartier (1996) J. Virol 70:4805-4810. This system uses a bacterial plasmid that contains a full length copy of an Ad genome coupled with a simple gene replacement method in E. coli. This allows manipulation of any portion of the Ad genome in a prokaryotic or eukaryotic expression vector followed by insertion into a full length copy of an Ad genome. The full length Ad chromosome is cut once with a restriction enzyme in the region one wishes to replace. Bacteria are co-transformed with this linearized molecule. Homologous recombination yields a circular molecule that is competent for replication in the bacterial cell. Presence of the altered Ad sequence can be confirmed by PCR and Southern blotting.
[0108] An encapsidated Ad is produced by transfecting the newly generated, mutated Ad genome into Ad-replication-competent cells, such as, e.g., 293 cell lines. This is followed by incubation, harvesting and plaque purification of the newly produced viruses.
[0109] When it is desired to isolate the viral particles from producer cells, the cells are lysed. A variety of means well known in the art can be used. For example, mammalian cells may be lysed under low pressure (100-200 psi differential pressure) conditions or conventional freeze thaw methods. Exogenous free DNA/RNA is removed by degradation with DNAse/RNAse. The viral particles (capsids) are then purified by means known in the art, e.g., chromatographic or differential density gradient. Viral particles can also be purified directly from the initial lysate by, e.g., CsCl density centrifugation, and mature virions collected from the gradient. In one exemplary protocol the treated, buffered cell lysate is first chromatographed over an anion exchange resin followed by chromatography over an immobilized metal affinity resin (see, e.g., U.S. Pat. No. 5,837,520). Anion-exchange high-performance liquid chromatography can also be used (see, e.g., Shabram (1997) Hum. Gene Ther. 8:453-465).
[0110] To confirm the genome was packaged (i.e., encapsidated) during the co-infection the lysate can be applied to a new culture of cells. About 48 hours later, viral DNA is isolated and Southern blot analysis is performed to assay for the presence of manipulated Ad sequence. If this sequence had been packaged during the first infection, it will be delivered into the cells during the second infection and amplified. Packaging can also be confirmed by purifying the virus on a density gradient and isolating and analyzing the DNA from the density purified virus. A capsid containing a complete genome can be identified by having a density greater than capsids only containing partial genome or no packaged nucleic acid. After capsid isolation based on density, DNA can be prepared from various capsid samples and analyzed or sequenced.
[0111] Growth of Adenovirus in Cell Culture
[0112] The vector systems of the invention are used to infect or transform adenovirus replication competent cells, such as 293 cell lines. The primary human embryonic kidney (HEK) 293 cell line is a permanent line of cells transformed by sheared human Ad 5 DNA. The cells are particularly sensitive to Ad and are highly permissive Ad DNA. This cell line is readily available from commercial sources, such as the American Type Culture Collection (ATCC CRL 1573). Variants of 293 cell lines can also be used, e.g., see U.S. Pat. No. 5,919,636.
[0113] Cells are grown under conditions and for sufficient periods of time to allow production of encapsidated vector. This is typically carried out in a standard cell culture or a bioreactor device for cell culturing. The design of the bioreactor should ensure sterility and provide for containment of the producer cell and recombinant virus. A variety of bioreactors are commercially available for the culture of anchorage dependent producer cells and suspension cultures and are well known to those of skill in the art. Bioreactors can be equipped with an agitation system to keep the contents uniformly mixed and to facilitate oxygen transfer and sensors that permit monitoring and manipulation of various parameters (e.g., temperature, pH, dissolved oxygen) to maintain growth within optimal ranges for cell growth. The cells can be grown in a bioreactor under perfusion conditions. In one exemplary protocol, the Ad replication competent “producer” cells are attached to microcarriers. Serum-free media is frequently used to growth Ad producer cells; this media can be supplemented with hormones such as insulin, transferrin, epidermal growth factor, or hydrocortisone. Serum free media may also be enhanced agents which upregulate or stabilize the viral binding receptors, such as the alpha v beta3 and alpha v beta5 integrins to improve infection efficiency. See, e.g., U.S. Pat. No. 5,994,134.
[0114] Characterization and Identification of Cis-acting Packaging Sequences
[0115] The invention provides adenovirus vectors comprising serotype specific cis-acting packaging sequences. Adenoviral genomes without a complete packaging sequence will not be inserted into a capsid structure. In wild type virus, the packaging sequence can contain up to five related domains, called “A repeats.” However, the A repeats are not completely functionally redundant; inactivation of individual repeats impairs packaging efficiencies of the resulting mutant viruses to different extents. The packaging sequence from the Ad5 serotype has been reported to be at the genomic left end at about nucleotides 194 to 380 (see Table 1 below). See, e.g., Grable (1990) supra; Grable (1992) supra; Schmid (1995) supra; Schmid (1997) supra; Schmid (1998) supra. Some groups have reported that at least three copies of A repeats are required for efficient DNA encapsidation (see, e.g., Grable (1990) supra; Grable (1992) supra). However, any amount of “A repeat sequence,” or “A repeat” efficiency or structural form, or variation of packaging domain sequence can be used in the vectors of the invention as long as they retain their ability to package nucleic acid into a completed Ad capsid in a serotype-matched manner with a 52/55 kDa trans-acting protein.
[0116] In wild type adenovirus the packaging signal maps at the left end of the Ad genome. Thus, in the design and construction of the vectors of the invention, this is one location for placement of a packaging sequence. However, in the vectors of the invention the packaging sequence can be placed in any position as long as it retains its ability to package nucleic acid into a completed Ad capsid in a serotype-matched manner with a 52/55 kDa trans-acting protein. For example, it has been noted that neither inversion of the Ad packaging domain nor its relocation to the right end of the genome terminus affected its function; however, it is reported that it must be located within 600 bases of the genomic terminus for proper function (see, e.g., Hammarskjold (1980) Cell 20:787-795; Hearing (1987) J. Virol. 61:2555-2558).
[0117] The vectors of the invention can use packaging sequence from any serotype, including the 47 distinct Ad serotypes known to infect humans (see above). Packaging signals can be identified by routine sequence identity and functional analysis, as described below. Identification of a functional packaging sequence from any serotype for use in the vectors of the invention can be routinely identified either by sequence identity (homology) to other serotypes and by functional assays.
[0118] For example, packaging region sequence identify between exemplary serotypes (using residue 192 to 400 of Ad5 as an exemplary packaging sequence) is set forth in Table 1, below. 1 TABLE 1 Serotype 3 4 5 7 1 1 4 2 7 0 3 1 8 6 9 3 7 4 00.0 9.2 8.1 9.0 9.5 7.2 0.4 4 1 7 8 5 7 4 00.0 0.2 9.3 1.4 8.4 0.1 5 1 6 4 7 4 00.0 8.3 4.5 0.3 0.4 7 1 3 7 3 00.0 8.8 6.2 8.7 1 1 3 4 2 00.0 4.0 5.2 1 1 5 7 00.0 0.0 4 1 0 00.0
[0119] One exemplary assay to identify whether a particular nucleic acid sequence e nucleic acid into an Ad capsid with a matched trans-acting element is described (1997) supra.
[0120] Characterization and Identification of 52/55 kDa Polypeptide Encoding Sequences
[0121] The invention provides adenovirus vectors comprising serotype specific trans-acting 52/55 kDa polypeptide-encoding sequences. The 52/55 kDa protein must be present in trans with the cis-acting packaging factor in the replication process to form mature virions. See, e.g., Gustin (1998) supra; K. Gustin, Ph.D. Thesis, University of Michigan, supra; Hasson (1989) J. Virol. 63:3612-3621.
[0122] The vectors of the invention can use 52/55 kDa protein-encoding sequence from any serotype, including the 47 distinct Ad serotypes known to infect humans. 52/55 kDa protein-encoding sequence can be identified by routine sequence identity to known (human or on-human) 52/55 kDa sequences, by functional analysis (as described below), and other known methods. Several 52/55 kDa protein encoding sequences are known, see, e.g., Genbank accession numbers: for Ad5 P04496; for Ad2: P03262; for Ad12: P36715; for Ad40: AAC13960; for Bovine Ad: AAD09721; for mouse Ad: AAB53752; for canine Ad: Q65948; for Fowl Ad: AAC54906; for ovine Ad AAA84973. Additional assays to identify whether a particular nucleic acid sequence encodes a functional, serotype specific 52/55 kDa polypeptide are described by, e.g., Gustin (1998) supra; K. Gustin, Ph.D. Thesis, University of Michigan, supra.
[0123] These routine assays are also used to determine if a 52/55 kDa polypeptide from one serotype can or cannot complement the encapsidation function of the cis-acting packaging sequence of another serotype. The invention is based on the new finding that a cis-acting packaging signal must be serotype matched with a trans-acting 52/55 kDa polypeptide. Typically, the cis- and trans-acting factors are from the same serotype. For example, an Ad5 cis-acting packaging sequence will be complemented by an Ad5 a trans-acting 52/55 kDa polypeptide but not (to a significant degree) by an Ad7 a trans-acting 52/55 kDa polypeptide. Thus, one exemplary vector system of the invention includes a gene therapy vector with an Ad7 cis-acting packaging signal and a helper virus with an Ad5 cis-acting packaging signal, with the system complemented by only an Ad7 a trans-acting 52/55 kDa polypeptide. However, as discussed above, a trans-acting 52/55 kDa polypeptide from another serotype may also be able to significantly complement the Ad5 cis-acting sequence. As long as the trans-acting factor from the third serotype cannot also complement (to a significant degree) the cis-acting sequence in the helper virus (the Ad7 cis-acting sequence in this example), it can be incorporated into the vector system of the invention. Thus, the vectors systems of the invention incorporate any polypeptide which has the serotype-specific activity of the desired a trans-acting 52/55 kDa polypeptide
[0124] In addition to the functional (encapsidation) assays, in vitro and cell-based in vivo assay systems can be used to screen for such the serotype-specific encapsidation activity, including, e.g., cis-acting packaging signal binding proteins. Relevant trans-acting factors from different serotypes can be identified in this manner. Many assays are available that screen for nucleic acid binding proteins and all can be adapted and used with for cis-acting packaging signals from various serotypes. For example, one means to identify a cis-acting packaging signal binding compound is by contacting a cis-acting sequence with a test compound and measuring the ability of the test compound to bind the selected nucleic acid. The test compound can be any agent capable of specifically binding to a cis-acting sequence, including compounds available in chemical (e.g., combinatorial) libraries, a cell extract, a nuclear extract, a protein or peptide. A variety of well-known techniques can be used to identify polypeptides which specifically bind to cis-acting sequences, e.g., mobility shift DNA-binding assays, methylation and uracil interference assays, DNase and hydroxy radical footprinting analysis, fluorescence polarization, and UV crosslinking or chemical cross-linkers. For a general overview, see, e.g., Ausubel (chapter 12, DNA-Protein Interactions). One technique for isolating co-associating proteins, including nucleic acid and DNA/RNA binding proteins, includes use of UV crosslinking or chemical cross-linkers, including e.g., cleavable cross-linkers dithiobis (succinimidylpropionate) and 3,3′-dithiobis (sulfosuccinimidyl-propionate); see, e.g., McLaughlin (1996) Am. J. Hum. Genet. 59:561-569; Tang (1996) Biochemistry 35:8216-8225; Lingner (1996) Proc. Natl. Acad. Sci. USA 93:10712; Chodosh (1986) Mol. Cell. Biol 6:4723-4733. Alternatively, cis-acting sequence-affinity columns can be generated to screen for potential binding proteins. In a variation of this assay, cis-acting sequence are biotinylated, reacted with a solution suspected of containing a binding protein, and then reacted with a strepavidin affinity column to isolate the nucleic acid or binding protein complex (see, e.g., Grabowski (1986) Science 233:1294-1299; Chodosh (1986) supra). The cis-acting sequence-binding protein can then be conventionally eluted and isolated. Mobility shift DNA-protein binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) is an extremely rapid and sensitive method for detecting specific polypeptide binding to DNA (see, e.g., Chodosh (1986) supra, Carthew (1985) Cell 43:439-448; Trejo (1997) J. Biol. Chem. 272:27411-27421; Bayliss (1997) Nucleic Acids Res. 25:3984-3990). Interference assays and DNase and hydroxy radical footprinting can also be used to identify specific residues in the nucleic acid protein-binding site, see, e.g., Bi (1997) J. Biol. Chem. 272:26562-26572; Karaoglu (1991) Nucleic Acids Res. 19:5293-5300. Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions, see, e.g., Lundblad (1996) Mol. Endocrinol. 10:607-612.
[0125] Proteins identified by these techniques can be further separated on the basis of their size, net surface charge, hydrophobicity and affinity for other ligands. In addition, antibodies raised against such proteins can be conjugated to column matrices and the proteins immunopurified. All of these general methods are well known in the art. See, e.g, Scopes, R. K., Protein Purification: Principles and Practice, 2nd ed., Springer Verlag, (1987).
[0126] Heterologous Nucleic Acids
[0127] The invention provides a replication defective adenovirus sequence comprising an Ad serotype-specific cis-acting packaging sequence and a heterologous nucleic acid, flanked by ITR sequences. The heterologous nucleic acid, as defined above, can comprise any non-viral nucleic acid sequence. When the heterologous nucleic acid is intended for in vivo administration, it can be designed, e.g., to encode therapeutic polypeptides or antisense sequences, such as cell toxins or apoptosis inducing agents to ablate specific cell targets, e.g., cancer cells.
[0128] Any size of heterologous nucleic acid can be used in place of wild type sequence. The adenoviral genome, which is a linear, double-stranded DNA, can be manipulated to accommodate heterologous (e.g., non-viral) genes that range in size, including reasonably large DNA inserts to about 38 kilobases (Bett (1993) J. Virol. 67:5911-5921). However, some upper and lower size limits may be necessary to effect packaging of the vector into an Ad capsid. Thus, as a general guideline, an upper limit for total vector size is about 105% of Ad wild type genomic length (see, e.g., Ghosh-Choudhury (1987) EMBO J. 6:1733-1739; Bett (1993) supra). Lower limits for total vector size have been suggested, see, e.g., Mitani (1995) Proc. Natl. Acad. Sci. USA 92:3854-3858. See also Alemany (1997) supra. It has been reported that a preferable total size of the DNA inserted into the Ad vector be is about 28 to 32 kilobases, possibly because Ad needs to have a minimum size to be stably propagated (Parks (1997) J. Virol. 71:3293-3298). If a “gutted” gene transfer vector is used (i.e., most or all of the Ad genes are deleted, as discussed above) and the size of the resulting vector, including heterologous gene insert, ITR, cis-acting packaging sequence and other sequences (e.g., promoters, cell specific enhancer sequences, hormone responsive elements, mammalian artificial chromosome elements or elements from autonomous replicating circular minichromosomes, and the like) is less than about 28-32 kb, it may preferable to include “stuffer DNA”. For example, the stuffer DNA may be DNA derived from prokaryotic or eukaryotic genomic noncoding regions. For pharmaceutical compositions, the stuffer DNA can be derived from noncoding human genomic DNA. It has also been reported that Ad vectors with a matrix association region (MAR) have higher expression levels (Sykes (1988) Mol. Gen. Genet. 212:301-309). Inclusion of a MAR sequence in the vector is believed to confer nuclear stability to the vector.
[0129] Heterologous nucleic acids can be operably linked to transcriptional control sequences, e.g., promoters, enhancers. Control sequences can comprise Ad sequences normally associated with wild-type Ad genome (e.g., adenovirus major late promoter, Ad MLP). Alternatively, heterologous control sequences can be employed where desired.
[0130] Useful heterologous promoter sequences include those derived from sequences encoding mammalian genes or viral genes, e.g., the SV40 early promoter, mouse mammary tumor virus LTR (MMTV LTR) promoter, a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter (e.g., the CMV immediate early promoter region), a Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. The vectors of the present invention can include selectable markers to, e.g., confer antibiotic resistance or sensitivity, impart color, change the antigenic characteristics when cells which have been transfected are grown in a selective medium. Selectable marker genes include, e.g., neomycin resistance gene (encoding aminoglycoside phosphotransferase) that allows selection in cells by conferring resistance to G418; the hygromycin-B resistance gene (encoding Hygromycin-B-phosphotransferase).
[0131] Exemplary heterologous nucleic acids used as transgenes in the vectors of the invention include, e.g., tumor suppressor genes, cyclin dependent kinase inhibitors, cytotoxic genes, cytostatic genes, proapoptotic genes, prodrug activating genes, tumor specific antigens, or antisense sequences. Examples of tumor suppressor genes include the retinoblastoma (Rb) gene and its variants Rb110 and Rb56, the MMAC-1 gene, the p53 gene, the gene, the NF-gene, p33, and p73. Cyclin dependent kinase inhibitors include, e.g., p27kip, p57kip2, p15ink4b, p18ink4c, p19ink4d, p16ink4a and p21sdi-1 genes. Cytotoxic genes are designed to have a toxic effect in the target cell, either alone or in conjunction with exogenous chemical agents (e.g. pro-drug activating genes). Examples include the cytotoxic domains of ricin, diphtheria, or Pseudomonas exotoxin as well as the adenovirus E3 11.6 gene, adenovirus E1a. Examples of pro-drug activating genes include the thymidine kinase and cytosine deaminase genes. Pro-apoptotic genes includes p53 and p53 pathway genes (e.g. bax, bid, caspases, cytochrome C, etc.) and adenovirus E4. Examples of other therapeutic transgenes that can be incorporated into the vectors of the invention include, e.g., interferons (alpha, beta, gamma and consensus), interleukins (e.g. IL-2, IL-4, L-10), or neurotransmitters (e.g., dopamine, serotonin, GABA, ACTH, NGF). In neuromuscular diseases, such as ALS or spinal cord injuries, a heterologous gene can be delivered by Ad vector to prevent cell death and promote survival. For example, adenovirus vectors containing a neurotrophin-3 gene can targeted delivery of NT-3 to motoneurons to treat ALS (see, e.g., Hasse (1998) J. Neurol. Sci. 160 Suppl 1:S97-105). Ad vectors can also be used to replace the mutated Duchenne muscular dystrophy gene (see, e.g., Hoffman (1999) Arch. Pathol. Lab. Med. 123:1050-1052). Ad vectors can be used to treat cystic fibrosis; the CFTR gene can correct defective Cl-transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors (see, e.g., Romanczuk (1999) Hum. Gene Ther. 10:2615-2626).
[0132] Formulation and Administration Pharmaceuticals
[0133] The invention also provides adenovirus vectors formulated as pharmaceuticals for the transfer of nucleic acids into cells in vitro or in vivo. In addition to dividing cells, adenovirus can be used to infect resting and nondividing cells, such as, e.g., dendritic cells and neurons (see, e.g., Zhong (1999) Eur. J. Immunol. 29:964-72; Miyaguchi (1999) Neuroreport 10:2349-2353); epicardial and pericardial tissue of a patient's heart (see, e.g., U.S. Pat. No. 5,797,870), and others. The vectors, vector systems and methods of the invention can be used to produce replication defective gene transfer and gene therapy vectors, particularly to transfer nucleic acids to human cells in vivo and in vitro. Using the vector system and methods of the invention, these sequences can be packaged as adenovirus gene therapy vector preparations that are substantially free of helper virus and used as pharmaceuticals in, e.g., gene replacement therapy (in somatic cells or germ tissues) or cancer treatment; see, e.g., Karpati (1999) Muscle Nerve 16:1141-1153; Crystal (1999) Cancer Chemother. Pharmacol. 43 Suppl:S90-9.
[0134] The vectors, vector systems, pharmaceutical compositions and methods of the invention can also be used in non-human systems. For example, human Ad 5 can be used in gene delivery in laboratory animals (e.g., mice, rats) as well as economically important animals (e.g., swine, cattle); see, e.g., Mayr (1999) Virology 263:496-506; Mittal (1996) Virology 222:299-309; Prevec (1990) J. Infect. Dis. 161:27-30.
[0135] These pharmaceuticals can be administered by any means in any appropriate formulation. Routine means to determine drug regimens and formulations to practice the methods of the invention are well described in the patent and scientific literature, and some illustrative examples are set forth below. For example, details on techniques for formulation, dosages, administration and the like are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.
[0136] Pharmaceutical Compositions
[0137] The invention provides a replication defective adenovirus preparation substantially free of helper virus with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. The pharmaceutical composition of the invention can further comprise other active agents, including other recombinant viruses, plasmids, naked DNA or pharmaceuticals (e.g., anticancer agents).
[0138] Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, e.g., to stabilize the composition or to increase or decrease the absorption of the agent and/or pharmaceutical composition. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of any co-administered agents, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition (see infra for exemplary detergents).
[0139] Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known, e.g., ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound depends, e.g., on the route of administration of the adenoviral preparation and on the particular physio-chemical characteristics of any co-administered agent.
[0140] The compositions for administration will commonly comprise a buffered solution comprising adenovirus in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. A variety of carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of capsids in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
[0141] Determining Dosing Regimens
[0142] The pharmaceutical formulations of the invention can be administered in a variety of unit dosage forms, depending upon the particular condition or disease, the general medical condition of each patient, the method of administration, and the like. In one embodiment, the concentration of capsids in the pharmaceutically acceptable excipient is between about 103 to about 1018 or between about 105 to about 1015 or between about 106 to about 1013 particles per mL in an aqueous solution. Details on dosages are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences; Sterman (1998) Hum. Gene Ther. 9:1083-1092; Smith (1997) Hum. Gene Ther. 8:943-954.
[0143] The exact amount and concentration of virus and the amount of formulation in a given dose, or the “therapeutically effective dose” is determined by the clinician, as discussed above. The dosage schedule, i.e., the “dosing regimen,” will depend upon a variety of factors, e.g., the stage and severity of the disease or condition to be treated by the gene therapy vector, and the general state of the patient's health, physical status, age and the like. The state of the art allows the clinician to determine the dosage regimen for each individual patient and, if appropriate, concurrent disease or condition treated. Adenovirus has been safely used for many years for human vaccines; see, e.g., Horwitz (1990) supra; Straus (1984) supra; Haj-Ahmad (1986) J. Virol., 57:267); Ballay (1985) EMBO, 4, 3861 (1985); PCT patent application WO 94/17832). Human adenoviruses have been used in humans as in vivo gene delivery vehicles (Graham & Prevec in New Approaches to Immunological Problems, Ellis (ed), Butterworth-Heinemann, Boston, Mass., pp. 363-390 (1992); Ragot (1993) Nature 361:647-650 (1993); Kozarsky (1993) Curr. Opin. Genet. Dev. 3:499-503); U.S. Pat. No. 5,981,225. These illustrative examples can also be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels administered when practicing the methods of the invention.
[0144] Single or multiple intrathecal administrations of adenoviral formulation can be administered, depending on the dosage and frequency as required and tolerated by the patient. Thus, one typical dosage for regional (e.g., IP or intrathecal) administration is between about 0.5 to about 50 mL of a formulation with about 1013 viral particles per mL. In an alternative embodiment, dosages are from about 5 mL to about 20 mL are used of a formulation with about 109 viral particles per mL. Lower dosages can be used, such as is between about 1 mL to about 5 mL of a formulation with about 106 viral particles per mL. Based on objective and subjective criteria, as discussed herein, any dosage can be used as required and tolerated by the patient.
[0145] The exact concentration of virus, the amount of formulation, and the frequency of administration can also be adjusted depending on the levels of in vivo (e.g., in situ) transgene expression and vector retention after an initial administration.
[0146] Routes of Delivery
[0147] The pharmaceutical compositions of the invention can be delivered by any means known in the art systemically (e.g., intravenously), regionally, or locally (e.g., intra- or peri-tumoral or intracystic injection, e.g., to treat bladder cancer) by, e.g., intraarterial, intratumoral, intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa), intra-tumoral (e.g., transdermal application or local injection). For example, intra-arterial injections can be used to have a “regional effect,” e.g., to focus on a specific organ (e.g., brain, liver, spleen, lungs). For example, intra-hepatic artery injection can be used if the anti-tumor regional effect is desired in the liver; or, intra-carotid artery injection. If it is desired to deliver the viral preparation to the brain, (e.g., for treatment of brain tumors), it is injected into a carotid artery or an artery of the carotid system of arteries (e.g., occipital artery, auricular artery, temporal artery, cerebral artery, maxillary artery, etc.).
[0148] The vectors of the present invention, alone or in combination with other suitable components can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer. Typically such administration is in an aqueous pharmacologically acceptable buffer as described above. Delivery to the lung can be also accomplished, e.g., by use of a bronchoscope. Gene therapy to the lung includes, e.g., gene replacement therapy for cystic fibrosis (using the cystic fibrosis transmembrane regulator gene) or for treatment of lung cancers or other respiratory conditions.
[0149] Additionally, the vectors employed in the present invention may be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas.
[0150] The pharmaceutical formulations of the invention can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
[0151] The adenoviral constructs can also be administered in a lipid formulation, more particularly either complexed with liposomes to for lipid/nucleic acid complexes (e.g., as described by Debs and Zhu (1993) WO 93/24640; Mannino (1988) supra; Rose, U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner (1987) supra) or encapsulated in liposomes, as in immunoliposomes directed to specific tumor markers. It will be appreciated that such lipid formulations can also be administered topically, systemically, or delivered via aerosol.
[0152] Kits
[0153] The invention provides kits that contain the vectors, vector systems or pharmaceutical compositions of the invention. The kits can also contain adenovirus replication-competent cells, such as 293 cells. The kit can contain instructional material teaching methodologies, e.g., means to isolate replication defective transgene containing adenovirus. Kits containing pharmaceutical preparations can include directions as to indications, dosages, routes and methods of administration, and the like.
[0154] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
EXAMPLES[0155] The following example is offered to illustrate, but not to limit the claimed invention.
Example 1[0156] Selective Packaging of Adenovirus Genome by Serotype Matching and Mismatching of Cis- and Trans-acting Packaging Factors
[0157] The following example demonstrates that the compositions and methods of the invention can be used to produce replication defective adenovirus vector preparations that are substantially free of replication competent “helper” virus. These novel compositions and methods are based on the finding that interaction between Ad cis-acting packaging regions and Ad trans-acting 52/55 kDa protein can be serotype matched to produce an encapsidated adenovirus. Typically, a 52/55 kDa protein from a given serotype supports encapsidation in conjunction with a packaging sequence from that same serotype.
[0158] In these experiments an adenovirus serotype 5 (Ad5) deletion mutant (specifically mutated in the 52/55 kDa polypeptide encoding region, called “pm8001”) was complemented in trans using 52/55 kDa proteins from a matching serotype. Adding the (serotype matched) Ad5 52/55 kDa protein in trans with pm8001 under replication permissive conditions allowed packaging of nucleic acid comprising Ad5 packaging sequence. However, 52/55 kDa protein from a different serotype (Ad7) failed to complement the pm8001 mutation.
[0159] Construction of a 52/55 kDa Protein Defective Adenovirus
[0160] To make a null mutant in the 52/55 kDa protein, base substitutions near the 5′ end of the 52/55 kDa open reading frame were made to create a series of stop codons. FIG. 1 shows the sequence of the N-terminus of the Ad5 52/55 kilodalton protein open reading frame (ORF) in upper case letters; directly below is the corresponding amino acid sequence. Below that in lower case are the point mutations that introduce a series of stop codons, indicated by asterisks.
[0161] Ad5 52/55 kilodalton protein ORFs with these mutations were built into both prokaryotic and eukaryotic expression vectors. It was confirmed that none of these vectors express any 52/55 kDa protein. The mutated sequence was then inserted back onto an adenovirus chromosome in a bacterial clone taking advantage of a bacterial recombination system as described by Chartier (1996) J. Virol 70:4805-4810. This system uses a bacterial plasmid that contains a full length copy of the Ad5 genome coupled with a simple gene replacement method in E. coli. This allows mutation of any portion of the adenoviral genome in a prokaryotic or eukaryotic expression vector followed by insertion of the mutation into a full length copy of the Ad5 genome. The adenovirus chromosome was cut once with a restriction enzyme in the region one wishes to replace. This was used to co-transform bacteria with a linear DNA molecule containing the engineered mutation. Homologous recombination yielded a circular molecule that was competent for replication in the bacterial cell. Presence of the mutation was confirmed by PCR and Southern blotting. A linear adenovirus was produced by transfecting the viral newly generated, mutated adenoviral genome into 293-L1 cells (293 cells that stably express the Ad5 52/55 kDa protein, see, Gustin (1998) J. Virol. 72:7860-7870) and incubation followed by harvesting and plaque purifying the newly produced viruses. This new 52/55 kilodalton protein null Ad5 virus mutant was designated “pm8001.”
[0162] The most striking phenotype of pm8001 virus was that it forms full length Ad genomes and fully formed, but empty, capsids (i.e., only protein, no inserted nucleic acid) in replication competent cells. The pm8001 mutant strain shows no packaging of nucleic acid into its capsids at all.
[0163] Complementation Experiments
[0164] Experiments showed that pm8001 can grow in 293 cells that constitutively express the wildtype Ad5 52/55 kilodalton protein but not in 293 cells alone or in cells complemented with a non-matching (Ad7) 52/55 kilodalton protein. Cells were infected with pm8001 alone, full length wildtype (wt) Ad5 alone, full length wt Ad7 alone, pm8001 plus Ad5, or pm8001 plus Ad7 using standard procedures. An additional control was to infect 293-L1 cells, which express the Ad5 52/55 kDa protein constitutively, with pm8001.
[0165] After 24 to 48 hours, enough time for the virus to complete its replication cycle, a viral lysate is prepared. Two approaches were taken to determine if the pm8001 genome was packaged (i.e., encapsidated) during the co-infection. In one approach, this lysate is applied to a new culture of 293 cells. 48 hours later, viral DNA is isolated and Southern blot analysis is performed to assay for the presence of pm8001 DNA. If this DNA had been packaged during the first infection, it will be delivered into the cells during the second infection and amplified. In the second approach, viral particles (capsids) are purified directly from the initial lysate by CsCl density centrifugation. Mature virion capsids are collected from the gradient. Capsids with full length Ad genomes will have greater densities than those with only partial genomes or no packaged nucleic acid. DNA is also prepared from these particles and assayed for the presence of pm8001 genomes.
[0166] DNA was extracted and digested with KpnI and SpeI, and Southern blot analysis performed. The mutation in pm8001 introduces an additional SpeI site into the Ad5 genome. Therefore, one of the higher MW restriction digest products (a “band” in a gel) that is present in the Ad5 digest sample is lost in the pm8001 digest with the concomitant appearance of two additional lower MW bands. The latter two bands are only present after SpeI digestion of the pm8001 genome (not the wt Ad5). Neither of these two bands (nor any of the other Ad5/pm8001-specific bands) were present in capsids generated from the wt Ad7 plus pm8001 co-transfected sample. Thus, wt Ad7 52/55 kDa protein could not substitute for the Ad5 protein and allow for (i.e., complement) packaging of the pm8001 genome.
[0167] These findings demonstrate that the cis- and trans-acting elements of the adenovirus genome packaging system must be serotype matched to produce a packaged adenoviral genome. Thus, the vector systems of the invention comprising adenoviral vectors with mismatched cis-acting packaging signals can be used to produce replication defective vector preparations (e.g., pharmaceuticals) substantially free of replication competent “helper” virus.
Example 2[0168] Selective Packaging of Adenovirus by Serotype Matching of Packaging Factors: Ad12 and Ad17 cannot complement Ad5
[0169] The following example further demonstrates that the compositions and methods of the invention can be used to produce replication defective adenovirus vector preparations that are substantially free of replication competent “helper” virus. These novel compositions and methods are based on the finding a 52/55 kDa protein from a given serotype is only able to support encapsidation in conjunction with a packaging sequence from that same serotype.
[0170] In the experiments described in Example 1, above, an Ad5 specifically mutated in the 52/55 kDa polypeptide encoding region (“pm8001”) complemented in trans using 52/55 kDa proteins from a matching Ad5 serotype; however, a non-matching Ad7 serotype could not complement the lack of trans-acting polypeptide. In Example 1, adding the (serotype matched) Ad5 52/55 kDa protein in trans with pm8001 under replication permissive conditions allowed packaging of nucleic acid comprising Ad5 packaging sequence; 52/55 kDa protein from Ad7 serotype could not complement the pm8001 mutation. Example 2's experiments demonstrated that adding serotype mis-matched Ad12 or Ad17 52/55 kDa protein in trans with pm8001 (lacking a 52/55 kDa protein) under replication permissive conditions also did not allow packaging of nucleic acid comprising Ad5 (pm8001) packaging sequence (i.e., could not complement the pm8001 mutation).
[0171] Using the same protocols as described in Example 1, human 293 cells or 293-L1 cells, which express the Ad5 L1 52/55 kDa protein, were infected with various virus(es), as indicated at the top of each lane of FIG. 2. “5” represents an Ad5 serotype, “12” represents an Ad12 serotype, “17” represents an Ad17 serotype. Ad17 was from ATCC catalog # VR-18; sequence is GenBank accession # NC—002067; Ad12 was from ATCC catalog # VR-863; GenBank accession # NC—001460.
[0172] After 48 hours, virions were purified from the infected cells by CsCl density centrifugation. Viral DNA was prepared from the virions, digested with KpnI+SpeI, and analyzed by Southern blotting with a mixed probe made from Ad5, Ad12, and Ad17 DNA. The arrows point to the two bands that are diagnostic of packaging of the pm8001 (&Dgr;L1) adenoviral DNA.
[0173] The data shows that neither Ad12 nor Ad17 can complement the pm8001 mutation (Ad5 serotype, but lacking a functional Ad5 52/55 kDa trans-acting protein). Thus, neither the Ad12- nor the Ad17-serotype 52/55 kDa trans-acting protein can effectively interact with Ad5 serotype cis-acting packaging sequences.
Example 3[0174] Ad7 Fails to Complement the Packaging Defect of pm8001
[0175] Since pm8001 requires the Ad5 52/55-kDa protein provided in trans to package its DNA, it allows us to investigate if the 52/55-kDa proteins from other serotypes can complement the pm8001 mutation. This was examined using co-infection experiments. Cells were infected with pm8001 alone or co-infected with pm8001 and wild type Ad7 or Ad5 at an MOI of 5 pfu/cell for each of the viruses. Forty-eight hours post infection, progeny virions were purified by CsCl density centrifugation. All infections except for pm8001 alone in 293 cells yielded particles that sedimented at 1.34 g/cm3, corresponding to mature virions. In addition, the Ad7 plus pm8001 infection and the pm8001 infection alone gave a strong band at 1.29 g/cm3, the density of empty capsids. When pm8001 was grown in 293-L1 cells, which stably express the Ad5 52/55-kDa protein, mature virions were produced. DNA was prepared from the virions isolated from the various infections and Southern blots were performed to determine if pm8001 DNA was packaged. DNAs from Ad7 and wild type Ad5 can be distinguished by their KpnI+SpeI restriction enzyme digestion patterns (FIG. 3B, lanes 6 and 7). In addition, the mutation in pm8001 generates an extra SpeI recognition site (FIG. 3A); therefore the mutant virus DNA can be distinguished from wild type Ad5 (FIG. 3B, lanes 2 and 6). As reported previously, pm8001-infected 293 cells yield undetectable packaged DNA (FIG. 3B, lane 1), while DNA is packaged into virions when the virus is grown in 293-L1 cells (FIG. 3B, lane 2). Co-infection of 293 cells with wild type Ad5 allowed pm8001 DNA to be packaged (FIG. 3B, lane 3). However, there was no detectable packaged pm8001 DNA in virions isolated from 293 cells co-infected with Ad7 (FIG. 3B, lane 4). Quantification of the results from multiple experiments indicates that the level of pm8001 is significantly less than 0.1% and often undetectable, as in FIG. 3. Mature viral particles from the co-infection with wild type Ad5 and Ad7 contained both Ad5 and Ad7 DNA, indicating that Ad7 does not inhibit wild type Ad5.
[0176] Serotypes Ad12 and Ad17 were also tested for their ability to complement pm8001 in a co-infection. The results were similar to the Ad7 co-infection; pm8001 viral DNA was not detected in the purified virions from the co-infected cells (FIG. 3C). However, when wild type Ad5 was co-infected with Ad17, the level of packaged Ad5 DNA was low, indicating that Ad17 may inhibit Ad5. These results indicate that packaging of adenovirus DNA may be serotype or subgroup specific.
Example 4[0177] Ad7 Fails to Inhibit pm8001 DNA Replication and Capsid Assembly in the Co-infected Cells
[0178] The absence of pm8001 DNA in virions isolated from 293 cells co-infected with other serotypes indicated that the Ad7, Ad12, or Ad17 52/55-kDa proteins could not complement the mutation in pm8001. However, inhibition of pm8001 DNA replication or capsid assembly by the other serotypes would also yield the same result. Since Ad7 did not inhibit wild type Ad5, in the following experiments we examined if Ad7 inhibits pm8001 DNA replication and capsid assembly. Viral DNA was extracted from Ad7 and pm8001 co-infected 293 cells at 12, 24, and 48 h post-infection, and Southern blots were performed to determine the extent of DNA replication at different time points. A co-infection with the two wild type viruses was performed as a control. The amount of DNA from both pm8001 and Ad7 increased as the infection proceeded, indicating that the mutant virus could replicate in the co-infected cells (FIG. 4A). To determine whether the two viruses replicated in parallel, the amount of radioactivity in the indicated bands A and B in FIG. 4A, which are Ad7- and Ad5/pm8001-specific respectively, was measured using a PhosphorImager. The ratio of the two bands, A to B, represents the ratio of replication of the two viruses at each time point. In the co-infections of Ad7 with either pm8001 or Ad5, similar ratios were found at all three time points. These data indicate that one virus in the co-infected cells did not affect the rate of replication of the other. The absolute amount of replication of pm8001 as compared to Ad5 was twofold lower.
[0179] To test if Ad7 blocks the assembly of pm8001 capsids, viral particles were isolated from the infected cells on CsCl gradient, and immunoblots were performed to detect the Ad5 IVa2 protein, which is present in both empty capsids and mature virions, using an Ad5 IVa2 protein-specific monoclonal antibody. As shown in FIG. 4C, this antibody recognizes the Ad5 IVa2 protein, but not the Ad7 IVa2 protein (lanes 4 and 5). The presence of Ad5 IVa2 protein in the empty capsids isolated from cells co-infected with pm8001 and Ad7 (lane 2) indicates that pm8001 empty capsids were assembled in the co-infected cells. Thus, the mutant virus replicated its DNA and capsids were formed in the co-infected cells, but the DNA was not encapsidated into progeny virions, demonstrating that Ad7 does not complement the packaging defect of pm8001.
Example 5[0180] Ad 5 and Ad7 Chimeric Virus
[0181] These co-infection experiments indicate that the Ad7 52/55-kDa protein cannot interact productively with the Ad5 packaging system in pm8001. To study this further, we constructed a chimeric virus, Ad7/5ITR&PSgr;-GFP, containing an Ad7 genome except for the ITRs and packaging sequence, which were derived from Ad5. In addition, the E1 region of Ad7 was replaced with a GFP expression cassette. The structure of this genome was confirmed by extensive restriction mapping and is shown in FIG. 5. Furthermore, the region containing the left ITR and &PSgr; was sequenced, and matched the Ad5 left ITR and packaging sequence.
[0182] Five micrograms of PmeI-digested pW120700 were used to transfect 293 cells. Viral DNA extracted from the transfected cells at various time points was digested with BclI. The input cosmid DNA produced in bacteria is dam-methylated, and should not be digested by BclI. BclI-digested replicated DNA was first detected at 1 d post-transfection, and increased at 3 d and 7 d (FIG. 6), indicating that the Ad7 E2 proteins were expressed in the transfected cells, and that these proteins functioned together with the Ad5 ITR to replicate the viral DNA.
[0183] Previous data indicated that the L1 52/55-kDa protein from Ad5 played a role in the growth of the chimeric virus. To examine this further, 293 cells or 293-L1 cells, which express the Ad5 L1 52/55-kDa protein, were transfected with PmeI-digested pW120700. FIG. 7 shows GFP expression in the transfected cells at 2 and 11 days post-transfection. Only single GFP-positive cells were detected at both time points, indicating that although viral DNA was replicating virus might not be spreading. Fourteen days post-transfection, CPE was not observed in either cell type. To confirm that viable virus was not being produced, viral lysates were made from the transfected cells at fourteen days post-transfection and used to infect fresh 293 cells. No GFP expression or CPE was detected in these cells, indicating that infectious virus was not produced in the initial transfection of 293 or 293-L1 cells, and that the presence of Ad5 52/55-kDa protein was necessary but not sufficient to allow packaging of the chimeric virus.
Example 6[0184] The IVa2 Protein Participates in Adenovirus Packaging
[0185] In order to identify additional proteins involved in adenovirus packaging, complementation assays involving the Ad5 IVa2 protein were performed. It was previously demonstrated that the IVa2 protein interacted with both the 52/55-kDa protein and the packaging sequence (Gustin et al., 1996, J. Virol. 70:6463). 293 or 293-L1 cells were co-transfected with PmeI-digested pW120700 and pBK-TripIVa2, which expresses the Ad5 IVa2 protein. FIG. 6 shows GFP expression in the co-transfected cells. In this experiment, in addition to single GFP-positive cells, clusters of GFP-positive cells were seen surrounding areas of CPE at nine to fourteen days post-transfection. This indicated that viruses were spreading from the area of the CPE to the surrounding cells. To confirm that infectious viruses were produced from the co-transfected cells, viral lysates were prepared 11-14 days after co-transfection and used to infect fresh 293 cells. GFP-expressing cells were found in these freshly infected 293 cells (FIG. 9A). Based on this result, a 293-cell line that stably expresses the Ad5 IVa2 protein was generated. The chimeric virus was able to spread on these cells (FIG. 9B, D, F). The virus did not grow any further in 293 cells (FIG. 9C and E), however, indicating that the AD5 IVa2 protein is required for the continuous growth of the chimeric virus.
[0186] This example further demonstrates that the compositions and methods of the invention can be used to produce replication defective adenovirus vector preparations that are substantially free of replication competent “helper” virus. These novel compositions and methods are based on the finding that interaction between Ad cis-acting packaging regions and Ad trans-acting IVa2 protein, a well as the 52/55 kDa protein, are involved in adenovirus encapsidation.
[0187] Plasmid Constructs
[0188] The entire ORF of the Ad5 IVa2 protein was amplified from the cDNA clone, E53, using these primers: 5′-GCGCGGATCCAAGATGGAAACCAGAGGGCGAAG-3′ and 5′-GCGCCTCGAGTT ATTTAGGGGTTTTGCG-3′. The PCR product was cloned into the BamHI and XhoI sites of pBK-CMV (Stratagene, La Jolla, Calif.) to generate pBK-IVa2. A cDNA of the Ad5 tripartite leader was cloned into the PstI and BamHI of pBK-CMV to generate pBK-Tripld. pBK-TripIVa2 was constructed by cloning the BamHI+XhoI-digested IVa2 fragment from pBK-IVa2 into the same sites of pBK-Tripld. pcDNA-TripIVa2 was generated by cloning the NheI+XbaI-digested IVa2 fragment from pBK-TripIVa2 into the same sites of pcDNA3.1/Hygro (Invitrogen).
[0189] Cells and Viruses
[0190] 293 cells are human embryonic kidney cells expressing adenovirus type 5 E1A and E1B proteins. 293-L1 cells are 293 cells that stably express the Ad5 52/55-kDa protein and are used as a helper cell line for growing the 52/55-kDa mutant virus, pm8001. Both these cell lines were maintained in DMEM with 10% FBS. For 293-L1 cells, 0.5 mg/ml G418 was added to the medium. A cell line that express the IVa2 protein was generated by co-transfecting 293 cell with pBK-TripIVa2 and pcDNA-TripIVa2. 10 &mgr;g of each plasmid was calcium phosphatate precipitated. 0.6 ml of the precipitate was added to 50% confluent 293 cells in a 60-mm dish. Fresh DMEM with 10% FBS and 500 &mgr;g/ml G418+100 &mgr;g/ml Hygromycin was added to the transfected cells 16 h later. When the cells became confluent, they were trypsinized, seeded at different cell concentration per dish and fed with medium containing 500 &mgr;g/ml G418 and 100 &mgr;g/ml Hygromycin. Individual colonies were selected and expanded. The expression of the IVa2 protein was detected by immunoblot. A cell line, which expressed the highest amount of the IVa2 protein was designated 293-IVa2.
[0191] Wild type Ad5, Ad7, Ad12, and Ad17 viruses (ATCC) were propagated on 293 cells. All infections were performed at an MOI of 5 pfu/cell; virus was allowed to adsorb for 2 h in DMEM with 2% FBS with gentle mixing every 15 min, followed by addition of DMEM with 10% FBS, and infected cells were harvested 48 h post-infection unless otherwise indicated.
[0192] Chimeric Virus Construction (Ad7/5ITR&PSgr;-GFP)
[0193] An Ad5 left end 384 bp DNA fragment containing the left ITR and packaging sequence (&PSgr;) was PCR-amplified using primers: 5′-GCGCATGCATGTTTAAACATCATCAATAATATACCTTA-3′ and 5′-GGCGGAGCTCACCTGGGCGAGTCTCCACGTA-3′. An Ad5 right end 200 bp fragment was amplified using primers: 5′-GCGCGGGCCCGTTTAAACATCATCA ATAATATACCTTA-3′ and 5′-GCGCGCATGCACAACTTCCTCAAATCGTCAC-3′. The PCR product of the left ITR+&PSgr; was cloned into the NsiI and SacI sites, and the right end ITR was cloned into the ApaI and SphI sites, of the pGEM-7Zf(+) vector (Promega) to generate pGEM-Ad5GV. Next, a Green Fluorescent Protein (GFP) expression cassette containing a CMV promoter, GFP ORF and SV40 poly (A) site was amplified from pEGFP-C1 (Clontech) and cloned into the SacI and BamHI sites of pGEM-Ad5GV to generate pGEM-Ad5GV-GFP. The fragment containing the left ITR-&PSgr;, the right ITR, and the GFP cassette in pGEM-Ad5GV-GFP can be released from the vector by PmeI digestion. The PmeI sites were added to the left and right ends of the left and right ITRs, respectively, during the PCR amplification.
[0194] Cloning of the Ad7 genome without the left most 2711 bp and right most 153 bp was accomplished by standard cloning and homologous recombination in E. coli. First, the Ad7 HindIII E fragment (nt 2712-6135 from the left end) was cloned into the HindIII site of pGEM-Ad5GV-GFP to generate pW111000. Then, the PmeI fragment in pW111000 containing the Ad5 ITRs and &PSgr;, the GFP expression cassette, and the Ad7 HindIII E fragment was cloned into the cosmid vector pWE15 (Clontech) to generate pW112700. An Ad7 right end 2.6 kbp fragment without the 153 bp right ITR was amplified by PCR using a primer from the 3′ end of the Ad7 fiber gene (5′-GCCCGGTACCTACACCAATCTCTCCCCACG-3′) and a primer just inside the Ad7 right ITR (5′-CGCGTCTAGATGACGTACCGTGAGAAA-3′). This fragment was cloned into the KpnI+XbaI sites of pW112700 to generate pW120100. The remainder of the Ad7 genome between these two fragments was introduced by recombination in E. coli (FIG. 5). 10 ng KpnI-digested pW120100 and 144 ng purified Ad7 viral DNA were co-transformed into E. coli BJ5183 cells. Colonies were screened by colony hybridization using 32P-labeled probes from the Ad7 sequence, which are not present in the parental pW120100. A positive clone was named pW120700, and contains the Ad5 ITRs and packaging sequence, a GFP expression cassette, and the entire Ad7 genome except for the left most 2711 bp and the right most 153 bp.
[0195] Viral DNA Isolation
[0196] Viral DNA was extracted from infected cells. DNA from CsCl gradient-purified virions was extracted by adding an equal volume of predigested pronase (2 mg/ml in 50 mM Tris, 1 mM EDTA, 0.5% SDS, pH 7.5) and incubating the mixture for 1 h at 37° C., followed by phenol extraction and ethanol precipitation. The DNAs were dissolved in TE.
[0197] Southern Blot and Immunoblot Analysis
[0198] For southern blots, DNA samples from 2.5×109 viral particles were digested with KpnI and SpeI, loaded on a 0.8% agarose gel for electrophoresis, then transferred to GeneScreen Plus hybridization membrane (NEN Life Science Products, Inc. Boston, Mass.). The membranes were prehybridized in hybridization buffer (1% SDS, 10% dextran sulfate, 1 M NaCl and 0.25 mg/mL of denatured sheared salmon sperm DNA) at 65° C. for 6 h before labeled probe was added. The 32P-labeled probe was generated by the Random Primer Labeling Kit (Life Technologies Inc., Gaithersburg, Md.) using both pTG3602, which contains the whole genome of Ad5, and purified Ad7, Ad12, or Ad17 genomic DNA as templates. 1×106 cpm/ml of the probe was added to the prehybridization buffer and incubated overnight at 65° C. The membrane was washed twice with 2×SSC (1×SSC is 0.15 M NaCl, 0.015M sodium citrate) at RT, then twice with 2×SSC, 1% SDS at 65° C. for 30 min. The membrane was dried and exposed to film. The intensities of the bands were measured by a PhosphorImager system (Molecular Dynamic Inc., Sunnyvale, Calif.). The limit of detection in this assay is 25 picograms viral DNA, the equivalent of 106 mature virions.
[0199] These data demonstrate that the invention's vector systems selectively package replication defective adenovirus nucleic acid sequences in an adenovirus capsid using a novel system based on mismatching of cis- and trans-acting adenovirus serotype packaging factors IVa2 and 52/55 kDa protein.
[0200] The present invention can be practiced by employing conventional materials, methodology and equipment. Accordingly, the details of such materials, equipment and methodology are not set forth herein in detail. In the previous descriptions, numerous specific details are set forth, such as specific vectors, cells, chemicals, processes, etc., in order to provide a thorough understanding of the present invention. However, it should be recognized that the present invention can be practiced without resorting to the details specifically set forth. In other instances, well known processing structures have not been described in detail, in order not to unnecessarily obscure the present invention. Only the embodiments of the present invention and but a few examples of its versatility are shown and described in the present disclosure. It is to be understood that the present invention is capable of use in various other combinations and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein. While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as come within known and customary practice within the art to which the invention pertains. Accordingly, other embodiments are within the scope of the following claims.
Claims
1. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs; and
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence.
2. The vector system of claim 1, wherein the adenovirus capsid, packaging and IVa2 trans-acting protein encoding sequences are human adenovirus sequences.
3. The vector system of claim 2, wherein the helper-dependent and helper adenovirus serotypes are selected from the group consisting of adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), and adenovirus type 40 (Ad40).
4. The vector system of claim 2, wherein the helper-dependent adenovirus serotype is adenovirus type 5 and the helper adenovirus serotype is adenovirus type 7.
5. The vector system of claim 2, wherein the helper-dependent adenovirus serotype is adenovirus type 7 and the helper adenovirus serotype is adenovirus type 5.
6. The vector system of claim 1, wherein the helper-dependent adenovirus sequence fails to produce a complete adenovirus capsid.
7. The vector system of claim 6, wherein the helper-dependent adenovirus sequence is encapsidated in a capsid comprising at least one polypeptide encoded by the helper adenovirus sequence.
8. The vector system of claim 6, wherein the helper-dependent adenovirus sequence is packaged in a capsid encoded by the helper adenovirus sequence.
9. The vector system of claim 1, further including a nucleic acid sequence encoding a 52/55 kDa protein, or a 52/55 kDa protein, wherein the polypeptide is from an adenovirus having the same serotype as the helper-dependent adenovirus serotype-specific cis-acting packaging sequence and different from the serotype of the helper adenovirus serotype-specific cis-acting packaging sequence.
10. The vector system of claim 1, further including a nucleic acid sequence encoding a 52/55 kDa protein, or a 52/55 kDa protein, wherein the polypeptide is from an adenovirus having a different serotype then the helper-dependent adenovirus serotype-specific cis-acting packaging sequence and different from the serotype of the helper adenovirus serotype-specific cis-acting packaging sequence.
11. The vector system of claim 1, wherein the failure to produce a functional IVa2 trans-acting protein is due to a mutation in the sequence encoding the protein.
12. The vector system of claim 11, wherein the mutation is a missense mutation, a point mutation, a frameshift mutation or a deletion mutation.
13. The vector system of claim 1, wherein the helper adenovirus sequence further comprises the nucleic acid sequence encoding the polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid.
14. The vector system of claim 1, wherein the nucleic acid encoding the polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is functionally-associated with the genome of an adenovirus replication competent host cell.
15. The vector system of claim 14, wherein adenovirus replication competent host cell is a 293 cell line.
16. The vector system of claim 1, wherein the polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is a IVa2 trans-acting protein.
17. The vector system of claim 1, wherein the helper-dependent adenovirus sequence lacks at least one nucleic acid sequence needed to produce a capsid and further comprises a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence.
18. The vector system of claim 1, wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, penton gene, fiber gene or hexon polypeptide gene or combination thereof.
19. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence,
- wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
20. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs; and
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (c) a cell comprising a nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein,
- wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
21. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs; and
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (c) an expression cassette comprising a nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein,
- wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
22. A vector comprising a replication defective adenovirus sequence comprising:
- (a) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence;
- (b) a nucleic acid sequence encoding a functional helper adenovirus serotype-specific IVa2 trans-acting protein, wherein the helper adenovirus serotype-specific IVa2 trans-acting protein does not have the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein, lacking the ability to produce a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein,
- wherein the replication defective adenovirus sequence comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
23. The vector of claim 22, further comprising at least one adenoviral nucleic acid sequence needed to produce an adenoviral capsid.
24. The vector of claim 23, further comprising sufficient adenoviral nucleic acid sequence to produce a complete adenoviral capsid when the vector is expressed in an adenovirus replication-competent host cell.
25. The vector of claim 22, wherein the helper-dependent and helper adenovirus serotypes are selected from the group consisting of adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), and adenovirus type 40 (Ad40).
26. The vector of claim 25, wherein the helper-dependent adenovirus serotype is adenovirus type 5 and the helper adenovirus serotype is adenovirus type 7.
27. The vector of claim 25, wherein the helper-dependent adenovirus serotype is adenovirus type 7 and the helper adenovirus serotype is adenovirus type 5.
28. A transformed or isolated infected cell comprising the vector system of claim 1 or the vector of claim 22.
29. A kit for making adenovirus encapsidated replication defective nucleic acid sequences comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs; and
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence.
30. The kit of claim 29, wherein the nucleic acid sequence encoding a polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is functionally-associated with the genome of an adenovirus replication competent host cell.
31. The kit of claim 29, wherein the nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein further comprises an expression cassette.
32. The kit of claim 29, wherein the helper adenovirus sequence further comprises the nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein.
33. A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps:
- (a) transforming or infecting into adenovirus replication competent host cells
- (i) a helper-dependent adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- a heterologous gene;
- (ii) a helper adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus ITRs;
- a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (iii) a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence; and
- (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.
34. A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps:
- (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences, wherein the cell comprises a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype IVa2 trans-acting protein that supports packaging of a helper-dependent adenovirus nucleic acid sequence and fails to support packaging of a helper adenovirus nucleic acid sequence,
- (i) a helper-dependent adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- a heterologous gene;
- (ii) a helper adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus ITRs;
- a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector, wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
35. A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps:
- (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences comprising:
- (i) a helper-dependent adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- a helper-dependent adenovirus serotype-specific cis-acting packaging sequence;
- a heterologous gene; and
- a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence; and
- (ii) a helper adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus ITRs;
- a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector, wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
36. The method of claim 33, 34, or 35 wherein the helper adenovirus sequence further comprises an adenoviral nucleic acid sequence encoding a complete adenoviral viral capsid.
37. A vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids, the vector comprising:
- (a) a replication defective adenovirus sequence comprising an adenovirus serotype 7 (Ad7) cis-acting packaging sequence;
- (b) a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 5 (Ad5) IVa2 trans-acting protein; and
- (c) an adenoviral nucleic acid sequence that encodes a viral capsid and fails to encode or produce a polypeptide having the activity of an adenovirus 7 serotype IVa2 trans-acting protein.
38. A pharmaceutical composition comprising an encapsidated replication defective adenovirus, made using the vector system of claim 1, substantially free of helper virus, and a pharmaceutically acceptable excipient.
39. The pharmaceutical composition of claim 38, wherein the pharmaceutical composition is 99% free of helper virus.
40. A method of delivering a heterologous nucleic acid to a cell comprising transforming or infecting a cell with the pharmaceutical composition of claim 38.
41. The method of claim 40, wherein the pharmaceutical composition is administered to a patient systemically, regionally or locally.
42. A packaging cell line for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the cell line comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and
- (c) a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence,
- wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
43. A packaging cell line for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the cell line comprising:
- (a) a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype-specific IVa2 trans-acting protein;
- (b) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);
- (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- (c) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs;
- (ii) a helper adenovirus serotype-specific cis-acting packaging sequence that fails to support the activity of the polypeptide having the activity of an adenovirus serotype-specific IVa2 trans-acting protein,
- wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.
44. A vector system for selectively packaging a replication defective nucleic acid sequence in a virus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ viral inverted terminal repeats (ITRs);
- (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid,
- wherein the helper-dependent adenovirus nucleic acid fails to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the first adenovirus serotype-specific cis-acting packaging sequence;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ virus ITRs;
- (ii) a second adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the second adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the second adenovirus serotype-specific cis-acting packaging sequence; and
- (c) a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific IVa2 trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence and a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific 52/55 kDa trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence.
45. A vector system for selectively packaging a replication defective nucleic acid sequence in a virus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ viral inverted terminal repeats (ITRs);
- (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid,
- wherein the helper-dependent adenovirus nucleic acid fails to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the first adenovirus serotype-specific cis-acting packaging sequence;
- (b) a helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ virus ITRs;
- (ii) a second adenovirus serotype-specific cis-acting packaging sequence,
- wherein the helper adenovirus nucleic acid fails to produce to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the second adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the second adenovirus serotype-specific cis-acting packaging sequence; and
- (c) a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific IVa2 trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence and a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific 52/55 kDa trans-acting protein that is derived from any adenovirus serotype that fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence.
46. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ ITRs from a first adenovirus serotype;
- (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;
- (b) a chimeric helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;
- (ii) a second adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,
- wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.
47. The vector system of claim 46, wherein the first adenovirus serotype is Ad7.
48. The vector system of claim 46, wherein the second adenovirus serotype is Ad5.
49. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ ITRs from a first adenovirus serotype;
- (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid fails to encode a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;
- (b) a chimeric helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;
- (ii) a second adenovirus serotype-specific cis-acting packaging sequence; and
- (iii) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,
- wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype; and
- (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype.
50. A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps:
- (a) transforming or infecting into an adenovirus replication competent host cell with adenovirus replication defective sequences comprising:
- (i) a helper-dependent adenovirus nucleic acid sequence comprising:
- 5′ and 3′ ITRs from a first adenovirus serotype;
- a first adenovirus serotype-specific cis-acting packaging sequence; and
- a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;
- (ii) a chimeric helper adenovirus nucleic acid sequence comprising:
- 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;
- a second adenovirus serotype-specific cis-acting packaging sequence; and
- an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,
- wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype; and
- (b) culturing the cells under conditions where the helper-dependent adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.
51. The method of claim 50, wherein the first adenovirus serotype is Ad7.
52. The method of claim 50, wherein the second adenovirus serotype is Ad5.
53. The method of claim 50, wherein the host cell is a 293 cell.
54. A chimeric helper adenovirus nucleic acid sequence comprising:
- (a) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;
- (b) a second adenovirus serotype-specific cis-acting packaging sequence; and
- (c) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,
- wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.
55. The chimeric helper adenovirus nucleic acid sequence of claim 54, wherein the non-second adenovirus serotype is Ad7.
56. The chimeric helper adenovirus nucleic acid sequence of claim 54, wherein the second adenovirus serotype is Ad5.
57. The chimeric helper adenovirus nucleic acid sequence of claim 54, wherein the nucleic acid is designated Ad7/5ITR&PSgr;-GFP.
58. A cell line for packaging the chimeric helper adenovirus nucleic acid sequence of claim 54, wherein the cell line comprises a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.
59. The cell line of claim 58, wherein the second adenovirus serotype is Ad5.
60. The cell line of claim 58, wherein the cell line is a 293 cell line.
61. The cell line of claim 58, wherein the cell line is designated 293-L1.
62. A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising:
- (a) a helper-dependent adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ ITRs from adenovirus serotype Ad7;
- (ii) an adenovirus serotype Ad7 cis-acting packaging sequence; and
- (iii) a heterologous nucleic acid;
- wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad7;
- (b) a chimeric helper adenovirus nucleic acid sequence comprising:
- (i) 5′ and 3′ adenovirus ITRs from adenovirus serotype Ad5;
- (ii) an adenovirus serotype Ad5 cis-acting packaging sequence; and
- (iii) an adenovirus serotype-specific nucleic acid sequence from adenovirus serotype AD7,
- wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad5.
Type: Application
Filed: Apr 29, 2002
Publication Date: Dec 12, 2002
Applicant: University of Michigan (Ann Arbor, MI)
Inventor: Michael J. Imperiale (Ann Arbor, MI)
Application Number: 10135895
International Classification: A61K048/00; C12N007/01; C12N015/861;
