Patent Application
20050003344
1. Technical Field
This invention relates to the field of molecular biology. In particular, the invention relates to methods and compositions of matter for promoting stable, site-specific integration of Rep-deleted recombinant adeno-associated virus (rAAV) vectors via delivery of a functional AAV Rep gene product to the necessary location by fusing it to an intercellular trafficking “cargo” protein such as herpes simplex virus (HSV) tegument protein, VP22 or fragment thereof.
2. Description of the Background Art
Recombinant adeno-associated virus vectors have recently emerged as promising vehicles for gene transfer for a variety of reasons, including their lack of pathogenicity, wide host range, ability to transduce nonproliferating target cells, stable genomic integration, and comparatively low intrinsic immunogenicity. Genetic and sequence analyses of wild type AAV2 have demonstrated two primary open reading frames (ORFs). The left ORF is necessary for virus DNA replication, and contains two promoters at map positions 5 (p5) and 19 (p19). These promoters control expression from colinear, overlapping reading frames that arise from unspliced and spliced transcripts which produce Rep proteins of 78, 68, 52, and 40 kDa respectively. The right ORF, which is necessary for virion encapsulation, contains a single promoter at map position 40 (p40), and encodes three overlapping proteins (VP1, VP2, and VP3) with alternative translational initiation sites. The AAV coding regions are flanked by inverted terminal repeats (ITRs) which possess weak intrinsic promoter activity and are critical for DNA replication, encapsulation and host cell integration. See Berns, in “The Parvoviridae: The Viruses and Their Replication,” Fields Virology, Fields, Knipe and Howley, Eds., 3rd edition, Lippincott-Raven, 1996, pp. 2173-2197; Chatterjee and Wong, “Adeno-associated virus vectors for transduction of genes encoding ribozymes,” in Intracellular Ribozyme Applications: Principles and Protocols, Rossi and Couture (Eds.), Horizon Scientific Press, 1999; Wong and Chatterjee, “Parvovirus Vectors for Cancer Gene Therapy,” in Cancer Gene Therapy, Lattine and Gershon, Eds., Academic Press, 2000.
One of the most interesting features of wild type AAV is its ability to integrate into a specific region in human chromosome 19 termed AAVS1. Kotin et al., Proc. Natl. Acad. Sci. USA, 87:2211-2215, 1990; Samulski et al., EMBO J. 10:3941-3950, 1991. Mutational and deletion analyses have demonstrated that this property is mediated by Rep68/78, the product of the p5 promoter. Surosky et al., J. Virol. 71(10):7951-7959, 1997. Theoretically, the capacity to integrate site-specifically would be highly advantageous for rAAV vectors for several reasons. From a safety standpoint, nonrandom integration would lessen the likelihood of insertional mutagenesis. Kung et al., Curr. Top. Microbiol. Immunol. 171:1-25, 1991. In addition, cellular sequence flanking inserts are known to affect trans gene expression, resulting in varying levels of expression depending upon the location of insertion. Lacy et al., Cell 34(2):343-358, 1983. Targeted vector integration could minimize this variability of expression.
The rep gene has been removed from essentially all currently used rAAV vectors, both to provide a larger space for insertion of recombinant transgenes and to minimize the risks of recombinational events generating wild type AAV during the encapsulation process. Thus, although some studies indicate that integration is not totally random, rep-minus, wild type free rAAV stocks no longer integrate site specifically into AAVS1. Fisher-Adams et al., Blood 88:492-504, 1996; Rivadeneira et al., Int. J. Oncol. 12(4):805-810, 1998.
There is a need in the art for methods to improve the potential safety of rAAV vectors and to modify gene expression from rAAV vectors, in particular, methods which would allow site specific integration of rep-deleted rAAV vectors. Delivery of a functional AAV rep gene product to the necessary location would be of great value in achieving safer gene transfer with less unpredictable expression levels. Restoration of site-specific integration of rAAV vectors could significantly impact upon the safety and utility of rAAV vectors for gene transfer and potential gene therapy.
Accordingly this invention provides a method for mediating site-specific integration of a rep-deleted rAAV vector in a cell, which comprises contacting the cell or expressing on the cell a fusion polypeptide which comprises an AAV2 Rep protein sequence of the left open reading frame of the rep gene that lacks a functional nuclear localization signal (NLS) and a VP22 polypeptide sequence that confers intercellular trafficking on the fusion polypeptide. The Rep protein may be fused at the carboxyl or amino terminus of the VP22 polypeptide and may be fused to it directly or indirectly, via a spacer of one or several amino acids. The AAV Rep protein preferably is truncated to remove amino acid residues 489, 490, 491 or 492 and the remaining carboxyl terminus of the translated Rep protein. The truncation most preferably is located at amino acid 490 or 491. DNA constructs and fusion proteins as described also form part of this invention. The invention also provides, in another embodiment, a method of increasing the level of integration of a rAAV vector in a cell comprising contacting the cell with a Rep fusion protein having a mutation in the AAV2 NLS.
Recently, a variety of peptides and proteins, such as the herpes simplex virus tegument protein VP22, have been shown to traffic intercellularly, both as native forms and as fusions with other proteins. See, for example, U.S. Pat. No. 6,251,398. This invention takes advantage of this ability to deliver a functional AAV gene product to cells to promote site specific RAAV integration and gene delivery.
Several peptides and proteins, collectively termed “cargo” proteins, which are capable of trafficking intercellularly have been described. These include the Drosophila antennaepedia protein, the HIV-1 tat protein and herpes simplex virus (HSV) tegument protein, VP22. See Schwarze and Dowdy, Trends Pharmacol. Sci. 21(2):45-48, 2000. Any known cargo protein is contemplated for use in the invention. Peptides and proteins fused in frame to these cargo proteins also are transported intercellularly, and, most importantly, can retain function. Intercellular transport and nuclear accumulation in vitro have been described with VP22 fused to green fluorescent protein (GFP), the tumor suppressor protein p53, and the herpes simplex virus thymidine kinase suicide gene. Elliott and O'Hare, Cell 88(2):223-233, 1997; Phelan et al., Nat. Biotechnol. 16(5):440-443, 1998; Dilber et al., Gene Ther. 6(1):12-21, 1999. Analogous studies have been reported for HIV-1 tat fusions with several cell cycle regulatory proteins, including p27Kipl and pl6lNK4a. Nagahara et al., Nat. Med. 4(12):1449-1452, 1998; Gius et al., Cancer Res. 59(11):2577-2580, 1999. P-galactosidase fused to HIV-1 tat trafficked widely in an in vivo mouse model. Schwarze et al., Science 285(5433):1569-1572, 1999). The exact mechanisms by which these proteins mediate intercellular transport have been difficult to elucidate, although transport mediated by HIV-1 tat appears to be receptor independent, and is more efficient when the tat fusion protein is denatured.
The ability of these cargo proteins to deliver functional genes was used in the present invention to promote site-specific rAAV integration and to increase the level of integration, to significantly enhance the potential safety of the gene delivery and to provide an improved method for expression. A variety of exemplary RepVP22 fusion constructs were constructed in which AAV rep or fragments thereof were linked in frame to the N- or C-terminus of VP22 within an expression plasmid (Invitrogen). These constructs were transfected into 293 cells, where protein expression, intercellular trafficking, and Rep function were monitored. These fusion constructs, for example AAV2Rep490VP22, VP22(Gly4)-AAV2Rep491 and VP22(Gly7)-AAV2Rep491, are considered part of the present invention. See Tables I and III-IV below and
Fusion constructs according to this invention are designed to traffic intercellularly by eliminating interference by the NLS present in AAV2 Rep. The AAV2 Rep NLS extends from amino acids 485-519 of the translated Rep ORF. A mutation in or truncation of the gene which deletes all or part of the NLS such that the NLS function is lost restores trafficking ability. Thus, according to the invention, genes truncated or otherwise mutated to remove the protein's ability to signal for nuclear localization are useful to deliver any desired gene and to promote high levels of site-specific integration of the gene and improve expression qualitatively and quantitatively. Therefore, any fusion polypeptide or DNA construct encoding such polypeptide having these properties may be used in the present invention.
Any polypeptide sequence that confers nuclear localization on the fusion polypeptide, as known in the art, may be used with the inventive compositions and methods. For example, VP22 polypeptides or fragments or variants thereof which retain the desired nuclear localization function are preferred. Other polypeptides suitable for use in these inventive fusion polypeptides include Drosophila antennaepedia protein, HIV-1 tat protein and functional fragments or variants thereof. Functional segments of the polypeptide, whether truncated at the carboxyl or amino terminus, or both or internal deletions are included in the term fragment. The term variant includes polypeptides containing amino acid substitutions, whether conservative or not, which are at least 80% homologous and preferably 90%, 95% or 99% homologous to the native sequence and which retain the desired nuclear localization function. Persons of skill in the art are well aware of methods of constructing or purifying such molecules and of manipulating them by molecular biological techniques to construct the desired DNA and protein fusions.
Rep protein sequences encoded by the left open reading frame of the AAV2 rep gene that lack a functional nuclear localization signal sequence are suitable for use with the invention. Any such Rep protein sequence may be used, including sequences having a mutation in the NLS which disturbs the NLS function sufficiently to restore trafficking ability. Persons of skill in the art are aware of known methods for determining whether this trafficking ability or the NLS function is present, absent, or sufficiently reduced to allow the inventive methods to operate in the system of choice, using known or routinely modified assays and other techniques. Therefore, any AAV2 Rep protein sequence in which NLS function is absent or severely curtailed (i.e. not detectable or at a level which does not interfere with the functioning of the inventive method) compared to the activity of full-length native Rep protein is contemplated for use with this invention.
Specifically, Rep protein sequences in which the NLS is deleted may be used, for example by deletion of amino acids 485-519 of the native sequence or by truncation of the carboxyl terminal portion of the Rep protein at amino acid 485, amino acid 486, amino acid 487, amino acid 488, amino acid 489, amino acid 490, amino acid 491, amino acid 492, amino acid 493 or amino acid 494. By truncation at an amino acid residue, it is indicated that the amino acids carboxyl terminal to the named amino acid are removed. For example, in a protein truncated at amino acid 491, the carboxyl terminal residue of such a protein would be amino acid 491. Any deletions of the NLS which disturb function as described above may be used. For example deletion of amino acids 485-519 or 486-518 or 489-492 are suitable. Persons of skill in the art consider it routine to construct a variety of such deletion mutants and/or truncations of proteins. Therefore, such variations are considered part of the inventive compositions and methods. Rep protein mutants having point mutations in the NLS also may be used, as well as Rep protein sequences in which all or part of the NLS sequence has been removed and replaced with non-functional spacer amino acid residues.
Western analysis demonstrated that all RepVP22 constructs were expressed as stable protein products of expected size. Full length rep fused to VP22 did not traffic intercellularly (data not shown). A fusion gene truncated at nucleotide 490 of the AAV2 rep gene sequence did traffic intercellularly as assessed by immunofluorescence microscopy and flow cytometry. See, for example,
A PCR assay which specifically detects vector integration into AAVS1, coupled with Southern analyses, suggested that all three constructs described in Table I promoted site specific rAAV2 vector integration. See
To confirm rAAV integration into the AAVS1 site by Rep490VP22, PCR products containing vector-cell junction fragments were cloned and sequenced. See
To exploit the ability of the fusion cargo proteins to deliver functional protein domains intercellularly, the wild type and several modified AAV2 Rep gene constructs were fused in frame to the VP22 ORF both in amino- and carboxyl-terminal orientations. The fusion proteins were expressed using the highly active CMV IE promoter. Although fusions of VP22 with full length AAV2 Rep did not appear to traffic, specific Rep fusion proteins in which the NLS was truncated, for example VP22(Gly7)-AAV2Rep491, trafficked intercellularly and were capable of promoting site specific integration of recombinant RAAV vectors. See
Fusion proteins according to the invention can be expressed by plasmid DNA transfection according to any method known in the art, including calcium phosphate coprecipitation, for example. Once expressed, the fusion proteins traffic to surrounding cells via the VP22 or other intercellular trafficking protein moiety, and can mediate rAAV vector site specific integration via the AAV Rep moiety. Those of ordinary skill in the art are familiar with such methods and are able to make modifications as desired depending on the protein fusion and cell type(s) involved. Alternatively, fusion proteins can be expressed within cells by introducing expression plasmid DNA via physical methods (lipofection, electroporation, etc.) or by using a viral vector. In addition, purified fusion protein may be applied directly to cells to promote site-specific rAAV integration. Because the constructs preferably express fusion proteins with His tags (which allow easy purification by nickel column chromatography) the proteins may be purified after production in bacteria or eukaryotic cells, and then applied directly to cells at the time of rAAV vector transduction. This increases the frequency of rAAV vector integration.
The references listed below are hereby incorporated into the specification by reference.
AAVS1 Site Specific Integration of rAAV.
Plasmids pVP22/myc-His and pVP22/myc-His-2 were obtained from Invitrogen (Carlsbad, Calif.). See
To construct a VP22-Rep fusion with the full length AAV2 Rep, see
The 5′-PCR primer used for the construction of the full length Rep-VP22 fusion was 5′GGTTTGAACGCGCAGATATCATGCCGGGG 3′ (SEQ ID NO: 3) which incorporated an EcoRV site (bold). Two different full length Rep-VP22 constructs were made: (1) RepVP22cys, in which the stop codon of Rep was modified to a Cysteine residue to allow read-through of VP22 and (2) RepVP22phe, in which amino acids 620 and 621 were eliminated and residue 619 was modified from a phenylalanine to a cysteine to allow for read-through of VP22. The downstream primer for the RepVP22cys construct was 5′GCCATACCTGATTTAGCGGCCGCATTGTTCAAAGATG 3′ (SEQ ID NO: 4), while the downstream primer used to generate RepVP22phe was 5′ GATTAAAATCATTTAGCGGCCGCAGATGCAGTCATCCAAA 3′ (SEQ ID NO: 5). Both primers incorporated a Not I site (bold) for cloning purposes. See
Progressive carboxyl terminal deletions into the AAV2 NLS were constructed using polymerase chain reaction and fused to the amino terminal portion (
The full-length AAV Rep fusion protein constructs were tested for their ability to traffic intercellularly as follows. 293 or COS cells were transfected with expression plamids encoding the fusion constructs and serially examined for spread of the fusion protein using indirect immunofluorescent microscopy after staining with an antibody directed against the myc tag common to all the fusion proteins. The constructs containing the full length AAV2 Rep did not traffic.
pVP22-Rep constructs with truncations in the NLS were constructed in a similar fashion to the previously described full length rep constructs. Rep proteins truncated at the carboxyl end at amino acids 484 (VP22AAVRep484), 491 (VP22AAVReP491), and 519 (VP22AAVRep519) were generated by PCR cloning. For these modified proteins, the 5′ end of the rep open reading frame was amplified with the same sense primer as VP22-Rep (5′GGGAGGTTTGATATCGCAGCCGCCATGCCGGGG 3′; SEQ ID NO: 1) and incorporated an EcoRV site (bold). The 3′ end of the Rep ORF for 484, 491 and 519 truncations were amplified with antisense primers, 5′ GGCTCCACCCTTTTTGTCTAGAAATTCATGCTCCAC 3′ (SEQ ID NO: 6), 5′ GGGGGCGGGTCTTTCTAGAGCTCCACCCTTTTTG 3′ (SEQ ID NO: 7), and 5′ GTTGATCGAAGCTTCTAGATCTGACGTCGATGG 3′ (SEQ ID NO: 8), respectively, all of which incorporated an Xba I site (bold).
For VP22(Gly)4AAVRep491 and VP22(Gly)7AAVReP491 constructs, the 5′ end of Rep ORF was amplified with 5′CCATTTTGAAGCGATATCGGTGGAGGCGGAGCCGCCATGCCGGGG 3′ (SEQ ID NO:9) and 5′ GGGTCTCCATTTGATATCGGGGGGGGTGGAGGCGGAGGCGCCATGCCGGGG 3′ (SEQ ID NO:10), respectively. EcoRV sites are in bold while bases encoding the glycine spacer residues are in bold and italicized. For the 3′ end, the antisense primer for the pVP22-Rep491 protein SEQ ID NO:7) was used. The amplified products were digested with EcoRV and Xbal, and inserted into similarly digested pV22/myc-His. Two full-length RepVP22 and three truncated RepVP22 constructs were generated.
Three truncated Rep constructs, AAVRep469VP22, AAVRep490VP22 and AAVRep505VP22, were created using independent Not I site-containing downstream primers coupled with the identical primer used to generate the full-length construct. The AAVRep469VP22 3′ primer, 5′GATCCTTTGCCCAGCGGCCGCCAGTCTTTGACTTCCTGCTTGG 3′(SEQ ID NO:11) extended from +1385 to +1425 with base changes at +1405 to +1408 and +1412. These sequence changes modified residue 469 from a phenylalanine to a cysteine and eliminated the production of all amino acids C-terminal to residue 469.
AAVRep490VP22 C-terminal primer, 5′GGTCTTTTGCGGCCGCCACCCTTTTTG 3′ (SEQ ID NO:12), extended from +1457 to +1483. Mismatches at +1469, +1471, and +1473 to +1475 were used to eliminate all residues C-terminal to 490. AAVRep505VP22 3′ primer, 5′GACTCGCGCACGCGGCCGCGCTCACTTATATCTGCG 3′ (SEQ ID NO:13), extended from +1496 to +1531. It contained nucleotide changes at positions +1513, +1515 to +1517 and +1520 resulting in the loss of amino acids C-terminal to residue 505. Additionally, residue 505 was modified from a proline to an arginine. All C-terminal primers above are given in the reverse orientation. Not I sites are indicated in bold.
The Rep protein sequence of the Rep491 truncated construct ends at amino acid 491 of the translated Rep protein, however there are 8 amino acids at the junction leading to the initiation codon of the VP22 polypeptide sequence. These amino acids (DIQHSGGR; SEQ ID NO:14) result from additional nucleotides found within the multiple cloning site. Therefore, it is clear to one of ordinary skill that multiple variations of the fusion peptides are possible, depending on the exact construction methods used to create them. The two moieties of the fusion polypeptide may be fused directly or indirectly, with additional amino acids present at the junction or either terminus. See Table II, below for exemplary sequences contained in the Rep fusion polypeptides compared to Rep wild type. All constructs were analyzed by DNA sequencing to insure that no additional mutations were inadvertently incorporated during the PCR amplifications. See Tables III-VI for sequence information for exemplary constructs.
All rep gene inserts were amplified using a PE 9600 thermal cycler (Perkin and Elmer). A standard 100 μl reaction contained 100 ng of template DNA, 25 pmol of each respective upstream and downstream primer, 2 units of Vent polymerase (New England Biolabs, Beverly, Mass.), 200 μM of each dNTP, 3 mM MgSO4, and 1× Vent reaction buffer. The mixture was denatured at 95° C. for 5 minutes, and then 25 cycles of amplification (95° C., 30 s; 60° C., 30 s; 72° C., 90 s) were performed, followed by one extension cycle at 72° C. for 7 minutes. PCR products were gel purified using the Prep-a-Gene™ purification kit (Bio-Rad Laboratories, Hercules, Calif.), digested with appropriate restriction enzymes (NEB) and ligated into corresponding vectors at 16° C. for 16 hours. Plasmid constructs were transformed into chemically competent DH5a cells using standard methods. Plasmids were purified by anion exchange column chromatography (Qiagen, Valencia, Calif.), and quantitated spectrophotometrically. Enzymes were used according to conditions suggested by the manufacturers. Oligonucleotides were synthesized using a 394 B DNA Synthesizer (Applied Biosystems, Foster City, Calif.). All constructs were sequenced to insure that mutations were not inadvertently introduced during amplification.
African green monkey Vero (#CCL-81), 293 cells, COS cells and a Detroit 6-derived cell line, 7374, which contains integrated wild type AAV2, were maintained in high glucose Dulbecco's MEM (DMEM) with 2 mM glutamine and 10% heat inactivated fetal calf serum, at 37° C. in 5% humidified CO2. All cells were routinely tested and found free of mycoplasma. All transfections were performed using a CellPhect Transfection kit (calcium phosphate procedure; Amersham Pharmacia, Piscataway, N.J.) according to the manufacture's directions. For Western blot of VP-Rep fusion proteins, 293 cells were transfected with VP22-Rep or Rep-VP22 constructs (or their associated modified constructs lacking a functional NLS) using calcium phosphate coprecipitation. Cells were harvested after 48 hours and lysed. Proteins were separated using SDS-PAGE electrophoresis, and transferred to nitrocellulose. The western analyses demonstrated expression of the AAVREP490V22 fusion protein following transfection. See
Rep Expression and Trafficking Analyses by Immunofluorescence Microscopy.
Amino and carboxyl-terminal VP22/AAV2 Rep fusion proteins encoded by expression plasmids were initially tested for their ability to traffic intercellularly after calcium-phosphate transfection into 293 cells. For immunofluorescence assays, approximately 6.0×105 293 cells were plated on coverslips in 6-well plates and transfected with 1-3 μg of expression vector DNA for the various Rep derivatives. At specified times post-transfection, cells were briefly washed 3 times in room temperature phosphate-buffered saline (PBS), fixed in methanol at −20° C. for 5 minutes, and permeabilized by incubating them in acetone for 2-5 minutes at −20° C. The fixed cells were subsequently blocked with 1% BSA/1× PBS for 5 minutes at room temperature and stained with 1 μM primary mouse monoclonal anti-rep (such as CAT# MAB6030, Maine Biotechnology, Portland, Me.) or anti-c-myc antibody (such as CAT# R950-25, Invitrogen) diluted in 1% BSA/1× PBS, for 1 hour. The cells were then washed 3 times in PBS, 5 minutes each time, and incubated for 1 hour with a FITC-conjugated goat anti-mouse IgG secondary antibody (such as CAT# sc-2010; Santa Cruz Biotechnology, Santa Cruz, Calif.) and DAPI (4′,6-diamidino-2-phenylindole; Sigma, St. Louis, Mo.). Following the washes, the fixed cells were briefly rinsed in sterile dH2O, air-dried, and mounted onto glass slides using a 50% glycerol in dH2O. All staining procedures were conducted at room temperature. Cells were photographed by epifluorescense on a Nikon Labophot-2 photomicroscope with fluorescein and DAPI filters using a Nikon Fluor 40× objective. No visible staining of the full length construct RepVP22 was seen outside of the nucleus. Therefore, it appears that Rep-NLS overrides VP22's inherent nature to traffic outside of cell.
The ability of VP22AAV2Rep491 and VP22(Gly)4AAV2Rep491 constructs to traffic intercellularly also were compared following transfection. Cells were stained for fusion protein with fluorescein isothiocyanate (FITC) and with 4′-6-diamidino-2-phenylindole-2HCl (DAPI) to visualize the cells. See
Flow Cytometry.
To further confirm intercellular transport of Rep-VP22 fusions, 293 cells were transfected with expression plasmids encoding the Rep-VP22 fusion proteins and analyzed by flow cytometry. See
Site-Specific Integration.
An rAAV vector-containing plasmid pCWRHIVAPAP and one of the relevant Rep derivatives were cotransfected using calcium phosphate into 1.8×106 293 cells seeded in 60 mm dishes. 293 cells were harvested between 60 and 90 hours post-transfection and washed twice with PBS at 4° C. Cell pellets were suspended in 100 mM NaCl, 25 mM EDTA, and 10 mM Tris, pH 8.0, with 1 μg/ml RNase A and incubated for 2 hours at 37° C. Sodium dodecyl sulfate (SDS) and Proteinase K then were added to a final concentration of 0.5% and 0.1 mg/ml, respectively, and the mixture was incubated overnight at 56° C. Genomic DNA was purified from the digested cell pellet material by phenol/chloroform extraction, followed by ammonium acetate/ethanol precipitation. Isolated DNA was quantified via spectrophotometric analysis. Similar experiments were performed using CWRHIVAPgkH, an rAAV vector encoding resistance to hygromycin. In these experiments, cells were grown in media supplemented with 250 μg/mL hygromycin and 400 μg/mL G418 to select for cells expressing the rAAV vector and fusion protein, respectively. Colonies resistant to both hygromycin and G418 were isolated and expanded. Genomic DNA was extracted from the cell lines as described above.
PCR analyses employing one primer within the vector and the other primer within AAVS1 were used to assess site-specific integration. Each 50 μl reaction contained 50 ng DNA template and 25 pmol each of a specific primer for rAAV vector and for AAVS1. Reaction mixtures were denatured at 95° C. for 5 minutes, cooled to 80° C. for 2 minutes (at which point the Taq polymerase was added), and then subjected to 35 cycles of amplification (94° C., 1 min; 55° C., 1 min; 72° C., 3 min), followed by a single extension cycle at 72° C. for 5 minutes.
To confirm rAAV vector site-specific integration into AAVS1, PCR products corresponding to vector cellular junction sequences were inserted into PGEM-T vectors (Promega, Madison, Wis.), amplified in DH5α cells, and subjected to agarose gel sequence analysis in two independent Southern analyses, one probed with an rAAV-specific (
rAAV-Cell Junction Sequence Analysis.
Preliminary DNA sequence alignment analyses of cell-vector junction sequences isolated following TA cloning of the junction fragment demonstrated both vector and AAVS1 sequences, indicating site-specific integration of the vector. See
This application claims the benefit of U.S. provisional application Ser. No. 60/432,258 filed Dec. 11, 2002.
This invention was made with government support in the form of grant no. HL60898-0lAl from the United States Department of Health and Human Services, National Institutes of Health, National Heart, Lung, and Blood Institute, and grant no. CA33572 from the Department of Health and Human Services, National Institutes of Health, the National Cancer Institute. The United States Government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60432258 | Dec 2002 | US |
