[0001] The therapeutic treatment of diseases and disorders by gene therapy involves the transfer and stable insertion of new genetic information into cells. Although a variety of physical and chemical methods have been developed for introducing exogenous DNA into eukaryotic cells, viruses have generally been proven to be more efficient for this purpose. Several DNA-containing viruses, such as parvoviruses, adenoviruses and herpesviruses, and RNA-containing viruses, such as retroviruses, have been used to construct eukaryotic cloning and expression vectors and explored as gene therapy vehicles.
[0002] Retrovirus- and adenovirus-based vectors are associated with certain complications and disadvantages. For example, retroviruses are intimately associated with neoplastic events. See Donahue, Helper virus induced T cell lymphoma in non-human primates after retroviral mediated gene transfer,
[0003] Thus, an alternative vector which is neither pathogenic nor immunogenic would be advantageous. In contrast to adenoviruses, the parvovirus, adeno-associated virus (AAV), has a much smaller genome, most of which can be replaced by foreign DNA. Parvoviruses are small, icohedral viruses approximately 25 nm in diameter containing a single strand DNA genome of approximately 5 kilobases (kb). They consist of two major classes: the dependoviruses, including AAV and its subtypes (AAV1, AAV2, AAV3, AAV4 and AAV5), and the autonomous parvoviruses. The latter lytically infect permissive, proliferating cells in nonintegrating manner without helper virus assistance. On the other hand, AAV is a non-pathogenic human parvovirus that requires co-infection with a helper virus, usually adenovirus (or herpesvirus), for its optimal replication. See for example, Berns, Parvovirus replication,
[0004] In the absence of a helper virus, the wild-type (wt) AAV has been shown to integrate into the human chromosome 19 in a site-specific manner. See Kotin and Berns, Organization of adeno-associated virus DNA in latently infected Detroit 6 cells,
[0005] A number of studies have reported AAV-mediated successful transduction and expression of therapeutic genes in vitro. For example, see Chatterjee, Dual target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector,
[0006] A few studies have examined the safety and efficacy of the AAV vectors in vivo (see Flotte, Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector,
[0007] A disadvantage of AAV vectors in some clinical indications is the generalized nature of AAV infection. Previous studies have indicated that AAV possesses a wide host range that transcends the species barrier. See for example Muzyczka, Use of adeno-associated virus as a general transduction vector for mammalian cells,
[0008] In one aspect, the invention provides methods for selectively expressing therapeutic molecules, such as secretory proteins, antisense molecules and ribozymes, in the liver. The methods find use in treating hepatic diseases or conditions. The methods also find use in treating any disease or condition in which systemic administration of the therapeutic substance, for example a secretory protein, is desired. The methods also find use in treating or diseases or conditions involving proteins that originate or are normally made in the liver.
[0009] The methods involve administering to a mammalian patient having a need for liver expression of a therapeutic molecule a therapeutically effective amount of an AAV vector containing a the therapeutic molecule. Therapeutic molecules useful in treating hepatic diseases or conditions which can be administered employing the methods described here include, for example, insulin and thymidine kinase,. Therapeutic molecules comprising proteins originating in the liver or protein normally made in the liver include, for example, the LDL receptor, Factor VIII, Factor IX, phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC), and α1-antitrypsin. Therapeutic molecules comprising secretory proteins in which systemic administration is advantageously attained via liver specific delivery include, for example, cytokines, growth factors and the colony stimulating factors, G-CSF and GM-CSF. Additional protein therapeutic molecules contemplated for use in the methods and compositions of the invention are described infra.
[0010] Also included are nucleic acid sequences that encode antisense molecules that are useful in treating a hepatic disease. The antisense molecule will be an RNA sequence that can prevent or limit the expression of over-produced, defective, or otherwise undesirable molecules by being sufficiently complementary in sequence to the target sequence that it binds to the target sequence. For example, the target sequence can be part of the mRNA that encodes a protein, and the antisense RNA would bind to the mRNA and prevent translation. The target sequence can be part of a gene that is essential for transcription, and the antisense RNA would bind to the gene segment and prevent or limit transcription. For example, Group C adenoviruses Ad2 and Ad5 have a 19 kiloDalton glycoprotein (gp 19) encoded in the E3 region of the virus that binds to class I MHC molecules in the endoplasmic reticulum of cells and prevents terminal glycosylation and translation of the molecule to the cell surface. Prior to liver transplantation, the liver cells may be infected with gp19-encoding AAV vectors or virions which upon expression of the gp 19 inhibit the surface expression of class I MHC transplantation antigens. These donor cells may be transplanted with low risk of graft rejection and may require a minimal immunosuppressive regimen for the patient. It may also permit a donor-recipient state to exist with fewer complications.
[0011] Similar treatments may be used to treat chronic hepatitis B infections or non-A non-B hepatitis. The vector can be engineered to include a structural hepatitis gene, polyadenylation signal or a fragment thereof in reverse orientation such that the expression product binds to hepatitis virus mRNA transcripts, preventing translation of the structural protein and ultimately “inactivating” the virus. See, for example, Wu, Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides,
[0012] Also included are nucleic acid sequences that encode ribozymes that are useful in treating various diseases and conditions. Ribozymes are RNA polynucleotides capable of catalyzing RNA cleavage at a specific sequence and hence useful for attacking particular mRNA molecules. In chronic myelogenous leukemia for example, the “Philadelphia chromosomal translocation” causes expression of a bcr-abl fusion protein and abnormal function of the abl oncoprotein. Because the fusion mRNA occurs only in cells that have undergone the chromosome tanslocation and because the fusion transcript contains only two possible sequences at the splice junction, a ribozyme specific for either of the two bcr-abl fusion mRNA splice junctions can inhibit expression of the oncoprotein. Exemplary ribozymes include ribozymes to hepatitis A, hepatitis B and hepatitis C. See Christoffersen and Marr,
[0013] Currently preferred therapeutic molecules are the LDL receptor, Factor VIII, Factor IX, PAH, TPO (thrombopoietin) and EPO (erythropoietin). Also preferred are growth factors and cytokines. A therapeutically effective amount of the therapeutic molecule for purposes of this invention is at least about 10
[0014] We contemplate that any AAV vector can be employed in the methods of this invention. Leading and preferred examples of such vectors for use in this invention are the AAV-2 basal vectors disclosed in Srivastava, PCT Patent Publication WO 93/09239. Most preferred are the vectors of the invention as disclosed herein. Such vectors comprise the two AAV ITRs (inverted terminal repeats) in which the authentic (i.e., native) D-sequences of the ITRs are modified by the substitution of nucleotides such that at least 5 authentic nucleotides and up to 18 authentic nucleotides, preferably at least 10 authentic nucleotides up to 18 authentic nucleotides, most preferably 10 authentic (i.e., native) nucleotides, are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native, i.e., exogenous nucleotides. One preferred sequence of 5 native nucleotides that are retained is 5′ CTCCA 3′. The authentic (i.e., native) D-sequences of the AAV ITRs are sequences of 20 consecutive nucleotides in each AAV ITR (i.e., there is one sequence at each end) which are not involved in HP formation. The exogenous or non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. For example, appropriate replacement nucleotides for native D-sequence nucleotide C are A, T and G and appropriate replacement nucleotides for native D-sequence nucleotide A are T, G and C. The construction of four such vectors is exemplified in Example 4, to wit, preferred vectors pD-5, pD-15 and pD-20, and most preferred vector pD-10, using the vector pXS-22 as starting material.
[0015] Other employable exemplary vectors are pWP-19, pWN-1 both of which are disclosed in Nahreini,
[0016] Although not an absolute requirement for the practice of the invention, in a further embodiment, the AAV vectors of the invention may contain a liver specific promoter to maximize the potential for liver specific expression of the exogenous DNA sequence contained in the vectors. The promoter is operably linked to the nucleic acid encoding the therapeutic molecule upstream from the latter and between the AAV vector sequences (for example between the inverted terminal repeats in psub201 or downstream of the Double D ITR sequence) Preferred liver specific promoters include the hepatitis B X-gene promoter and the hepatitis B core protein promoter. These liver specific promoters are preferably employed with their respective enhancers. The enhancer element can be linked at either the 5′ or the 3′ end of the nucleic acid encoding the therapeutic molecule. The hepatitis B X gene promoter and its enhancer can be obtained from the viral genome as a 332 base pair EcoRV-NcoI DNA fragment employing the methods described in Twu,
[0017] We also contemplate that any hepatic disease or any defect in hepatic function, whether inherited or acquired, is susceptible to treatment with the methods of the invention. Exemplary hepatic diseases or defects in hepatic function include hepatocellular carcinoma, jaundice, infectious hepatitis, alcohol liver damage, including alcohol induced cirrhosis, and non-alcohol induced liver cirrhosis.
[0018] We also contemplate that any inherited or acquired disease or defect, the treatment of which requires administration of a therapeutic molecule that is normally made in the liver, is susceptible to treatment with the methods of the invention. Exemplary inherited diseases include familial hypercholesterolemia, which is caused by an LDL receptor deficiency, phenylketonuria, which is caused by a phenylalanine hydroxylase deficiency, urea cycle disorders, organic acid disorders, Wilson's disease, tyrosinemia, α
[0019] We also contemplate that the methods described here find use in treating any disease or condition in which the therapeutic substance, for example a secretory protein, is advantageously expressed in the liver in order to, for example, obtain systemic administration via entry into the circulatory system through the hepatic system. Genes encoding any of the cytokines and immunomodulatory proteins described here can be expressed in an AAV vector to achieve liver specific in vivo expression. Forms of these cytokines other than the forms mentioned here that are known to the skilled artisan can be used. For instance, nucleic acid sequences encoding native IL-2 (interleukin 2) and γ-interferon can be obtained as described in U.S. Pat. Nos. 4,738,927 and 5,326,859 respectively, while useful mutants of these proteins can be obtained as described in U.S. Pat. No. 4,853,332. As an additional example, nucleic acid sequences encoding the short and long forms of M-CSF (macrophage colony stimulating factor) can be obtained as described in U.S. Pat. Nos. 4,847,201 and 4,879,227 respectively. AAV vectors expressing cytokine or immunomodulatory genes can be produced as described here.
[0020] AAV vectors producing a variety of known polypeptide hormones and growth factors can be used in the methods of the invention to produce therapeutic expression of these proteins. Some such hormones, growth factors and other proteins are described in EP patent 0 437 478 B1 for instance. Nucleic acid sequences encoding a variety of hormones can be employed, including for example, human growth hormone, insulin, calcitonin, prolactin, follicle stimulating hormone, luteinizing hormone, human chorionic gonadotropin, thyroid stimulating hormone. AAV vectors expressing polypeptide hormones and growth factors can be prepared by methods known to those of skill in the art. As an additional example, nucleic acid sequences encoding different forms of human insulin can be isolated as described in EP patent publication 026598 or 070632 and incorporated into AAV vectors as described here.
[0021] Any of the polypeptide growth factors can also be administered therapeutically by liver specific expression in vivo with an AAV vector. For instance, different forms of IGF-1 and IGF-2 growth factor polypeptides are well known in the art and can be incorporated into AAV vectors for liver specific expression. See EP patent 0 123 228 B1. Liver specific expression of different forms of fibroblast growth factor can also be effected by the methods of the invention. See U.S. Pat. Nos. 5,464,774; 5,155,214 and 4,994,559.
[0022] There are a number of proteins useful for treating hereditary disorders that can be expressed by the methods of the invention. Many genetic diseases caused by inheritance of defective genes result in the failure to produce normal gene products, for example, severe combined immunodeficiency (SCID), hemophilia A, hemophilia B, adenine deaminase deficiency, Gaucher's syndrome, hereditary lactose intolerance and inherited emphysema. Also contemplated are diseases that are caused by the inability of the gene to produce adequate levels of the appropriate hormone, such as diabetes and hypopituitarism.
[0023] Liver specific expression of Factor VIII or Factor IX, useful for the treatment of blood clotting disorders such a hemophilia, is obtainable using the methods of the invention. PCT Patent Publication WO 96/21014 describes Factor VIII and HGH (human growth hormone) constructs for retroviral expression which could readily adapted by the skilled artisan for AAV expression. The Factor VIII minigene (see EP Patent Publication 232 112 and PCT Patent Publication WO 91/07490) could advantageously be employed for AAV expression. Also contemplated is the expression of lactase for the treatment of hereditary lactose intolerance, ADA for the treatment of ADA deficiency and α-1 antitrypsin for the treatment of α-1 antitrypsin deficiency. See Ledley,
[0024] There are a variety of other proteins of therapeutic interest that can be expressed in a liver specific manner using the methods of the invention. For instance sustained expression of tissue factor inhibitory protein (TFPI) is useful for the treatment of conditions including sepsis and DIC and in preventing reperfusion injury. See PCT Patent Publications WO 93/24143, WO 93/25230 and WO 96/06637. Nucleic acid sequences encoding various forms of TFPI can be obtained, for example, as described in U.S. Pat. Nos. 4,966,852; 5,106,833 and 5,466,783, and can be incorporated into AAV vectors as described here.
[0025] Other proteins of therapeutic interest such as erythropoietin (EPO) and leptin can be expressed in the liver by AAV vectors according to the methods of the invention. EPO is useful in gene therapy treatment of a variety of disorders including anemia. See PCT Patent Publication WO 95/13376. Gene therapy delivery of the leptin gene and its use in the treatment of obesity is described in PCT Patent Publication WO 96/05309. AAV vectors expressing EPO or leptin can readily be produced and liver specific expression attained employing the described methods. Other exemplary proteins and polypeptides include the cytokines such as interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14 and IL-15, α-interferon, β-interferon, the γ-interferons, GM-CSF, the tumor necrosis factors (TNFs), CD3, ICAM-1, LFA-1, LFA-3, the chemokines including RANTES 1α, MIP-1α, MIP-1β (see Cocchi, Science 720 (1996) 1811-1815) or analogs of such proteins. Because soluble forms of receptors can often behave as antagonists, as can mutated forms of the factors themselves, the nucleic acid sequences of therapeutic interest may also be agonists, antagonists or ligands for these proteins and polypeptides.
[0026] Even more proteins and polypeptides of therapeutic interest that can be expressed in liver specific fashion employing the AAV vectors and methods of the invention include Protein S and Gas6, thrombin, Coagulation Factor Xa, CSF-1 or M-CSF, IGF-1, IGF-2, acidic FGF, basic FGF, keratinocyte growth factor (KGF), TGF, platelet derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and HGF activators, PSA, nerve cell growth factor (NCGF), glial cell derived nerve growth factor (GDNF), VEGF, Arg-vasopressin, thyroid hormones, azoxymethane, triiodothyronine, LIF, amphiregulin, soluble thrombomodulin, stem cell factor, osteogenic protein 1, the bone morphogenic proteins, MGF, MGSA, heregulins and melanotropin. Growth factors can also be used in combination with mixtures consisting of one or several of, for example, DGF, IGF, PDGF, FGF or KGF. The full length growth factor can be employed or forms of the growth factor, such as active fragments, truncated forms and analogues can be employed. By “active fragment” we mean a polypeptide containing less than a full-length sequence that retains sufficient biological activity to be used in the methods of the invention. By “analogue” we mean truncated forms, splice variants, variants with amino acid substitutions, deletions or additions, alleles and derivatives of the mature protein or polypeptide which possess one or more of the native bioactivities of the full length protein or polypeptide. Thus, polypeptides that are identical or contain at least 60%, preferably, 70%, more preferably 80% and most preferably 90% amino acid sequence homology to the amino acid sequence of the mature protein wherever derived, from human or non-human sources are included within this definition. For example, a preferred truncated form of KGF is described in PCT Patent Publication WO 95/10434. See also PCT Patent Publication WO 90/08771 and U.S. Pat. No. 5,096,825 relating to human EGF.
[0027] The growth factor polypeptides, fragments and analogues can be produced by isolation from naturally occurring sources, polypeptide chain synthesis by peptide synthesis methods and production or recombinant proteins. These methods are well known to those of skill in the art. For example, production of recombinant PDGF is described in U.S. Pat. Nos. 5,045,633 and 4,769,328 and production of recombinant FGF and analogues is described in U.S. Pat. Nos. 5,229,501; 5,331,095 and 5,143,829.
[0028] A variety of other disorders can be treated by the methods of the invention. For example, production of apolipoprotein E or apolipoprotein A, useful in treating hyperlipidemia, can be attained via administration of the liver specific AAV vectors of the invention. See Breslow,
[0029] Nucleic acid sequences that encode the above-described proteins and polypeptides are obtainable from a variety of sources. For example, plasmids containing sequences the encode altered cellular products may be obtained form a depository such as the American Type Culture Collection (ATCC, Rockville, Md.) or from commercial sources such as Advanced Biotechnologies (Columbia, Md.) and British Bio-Technology Limited (Cowley, Oxford, Great Britain). Exemplary plasmids include ATCC Nos. 41000 and 41049 containing muteins of ras. Other nucleic acid sequences that encode the above-described proteins and polypeptides, as well as other nucleic acid molecules such as antisense sequences and ribozymes that are advantageously used in the invention may be readily obtained from such public sources. Exemplary are BBG12 containing the full length GM-CSF coding sequence, BBG6 containing the γ-interferon coding sequence, ATCC No. 39656 containing sequences encoding TNF, ATCC No. 20663 containing sequences encoding α-interferon, ATCC Nos. 31902 and 39517 containing sequences encoding β-interferon, ATCC No. 67024 containing the interleukin-1b coding sequence, ATCC Nos. 39405, 39452, 39516, 39626 and 39673 containing sequences encoding interleukin-2, ATCC No. 57592 containing sequences encoding interleukin-4, ATCC Nos. 59394 and 59395 containing sequences encoding interleukin-5 and ATCC 67153 containing sequences encoding interleukin-6. Molecularly cloned genomes encoding the hepatitis B virus are obtainable from the ATCC. ATCC No. 45020 contains the total genomic DNA of hepatitis B (with correctable errors), extracted from purified Dane particles, in the BamHI site of pBR322. See Blum
[0030] In another embodiment, AAV hybrid (i.e., chimeric) vectors are provided containing the DNA sequence, or functional fragment thereof, encoding hepatitis B surface antigen and the DNA sequence encoding the AAV capsid protein. An oligonucleotide sequence that corresponds to this HBV surface antigen peptide is blunt-ended and ligated at the 5′ end of the AAV VP-1 gene. Specifically, the 27 amino acid sequence of HBV surface antigen corresponding to amino acids 20-47 of the preS1 region (see, Ishikawa,
[0031] To establish integration of the vector into the chromosome of a host cell, host cells are transfected with the vector or infected with mature virions containing the vector. Methods of transfection are well-known in the art and include, for example, naked DNA transfection, microinjection and cell fusion. Virions can be produced by coinfection with helper virus such as adenovirus, herpes virus or vaccinia virus. Following coinfection with the vector and a helper virus, the host cells are isolated and the helper virus is inactivated. The resulting helper free stocks of virions are used to infect host cells. Alternatively, virions are produced by cotransfecting helper virus-infected cells with the vector and a helper plasmid. The plasmid will contain the parvovirus rep gene and non-AAV ITRs, for example adenovirus ITRs. Following cotransfection, mature virions are isolated using standard methods and any contaminating adenovirus inactivated using methods known to skilled artisans. The resulting mature virions can be used to infect host cells in the absence of helper virus.
[0032] Methods of making recombinant AAV vectors and packaging cell lines, purification methods, rescue methods and methods of generating high-titer vector stocks are known in the art. See for example, Samulski, A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication,
[0033] The vector or virions can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly humans. The vector or virions can be formulated in nontoxic, inert, pharmaceutically acceptable aqueous carriers, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8. Such sterile compositions will comprise the vector or virion containing the nucleic acid encoding the therapeutic molecule dissolved in an aqueous buffer having an acceptable pH upon reconstitution. Such formulations comprise a therapeutically effective amount of a AAV vector or virion in admixture with a pharmaceutically acceptable carrier and/or excipient, for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars and the like are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, this formulation is stable for at least six months at 4° C.
[0034] The virions can be systemically administered by intravenous injection. The dosage regimen will be determined by the attending physician or veterinarian considering various factors known to modify the action of drugs such as, for example, the physical condition of the patient, the severity of the condition, body weight, sex, diet, time of administration and other clinical factors. Generally, the regimen should be in the range of about 10
[0035] The AAV vector or virions can also be administered ex vivo employing art recognized methods, for example, by electroporation following the procedures of Chakrabarti,
[0036] The liver specific delivery methods of the invention may be employed with or without pretreatment of the liver. Pretreatment includes benign hyperplasia, which can be induced by treatment with HGF and/or transforming growth factorα. See Lui,
[0037] In another embodiment of the invention the AAV vector is co-administered with a cholesterol lowering drug to a primate patient suffering from hypercholesterolemia. A preferred cholesterol lowering drug is M-CSF. See U.S. Pat. Nos. 5,021,239 and 5,019,381. Other preferred cholesterol lowering drugs include niacin, gemfibrozil, lovastatin and mevacor.
[0038]
[0039]
[0040]
[0041]
[0042] In murine mammalian patients the fate of AAV vectors was followed after direct intravenous injection and it was surprisingly found that the AAV vectors possess organ-tropism for liver. Our AAV vectors contained the lacZ reporter gene or the human globin gene. In mice administered the lacZ reporter gene containing AAV vectors, expression occured in hepatocytes but a cytotoxic T lymphocyte response against βGal was not detected. The recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells.
[0043] The AAV recombinant virus stocks containing the CMV promoter (CMV
[0044] These highly purified recombinant AAV vectors were administered to C57B1/6 mice by direct intravenous injection into the tail vein.
[0045] Highly purified recombinant AAV vectors containing the cytomegalovirus (CMV) promoter-driven lacZ gene (vCMVp-lacZ) were directely injected into C57B1/6 mice. Approximately 1×10
[0046] Approximately 1×10
[0047] The results of the Southern blot analysis are shown in
[0048] The results in Example 1 were corroborated by injecting recombinant vHS2-βp-
[0049] Highly purified recombinant AAV vectors containing the human β-globin promoter-driven human
[0050] Approximately 1×10
[0051] We then investigated copy number of the vHS2-βp-
[0052] We next examined whether the lacZ gene delivered by direct injection of the r-AAV was transcriptionally active. Livers from mock-injected and vCMVp-lacZ-injected C57B1/6 mice were obtained one week p.i., and cryopreserved. Tissue sections were fixed and stained with 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (XGal) as described in Cheng, Separable Regulatory Elements Governing Myogenin Transcription in Mouse Embryogenesis,
[0053] Livers were obtained one week p.i. and frozen immediately in iso-pentane at −40° C. Sections of 15 μm were prepared using a cryostat and fixed in a solution containing 2% formaldehyde/0.2% para-formaldehyde in phosphate-buffered saline (PBS, 135 mM NaCl/2.5 mM KCl/8 mM Na
[0054] Co-transfection of an rAAV vector containing the AAV ITRs and the nucleic acid sequence encoding a therapeutic molecule and a helper plasmid containing the necessary rep and cap functions into adenovirus-2 (Ad2) infected 293 cells was expected to eliminate homologous recombination events leading to the production of contaminating wild-type (wt) AAV during the production of recombinant vector stocks. However, contaminating “wild type-like AAV” particles have been observed in such stocks ranging from 0.1% to 10%.
[0055] To determine the mechanism of generation of contaminating wt AAV, stocks were amplified through four successive round of co-infection with Ad2 in 293 cells. Low molecular weight DNA fragments were isolated, digested with Bal I restriction endonuclease and molecularly cloned into a pBlueScript plasmid vector. AAV sequence-positive clones were subjected to nucleotide sequencing using T3 and T7 primers. Nucleotide sequence analysis of 12 independent clones revealed that most of the recombination events leading to the contaminating wt AAV involved 10 nucleotides in the AAV D-sequence distal to viral hairpin structures. In addition, by analyzing 22 different clones generated with a helper plasmid that lacks the Ad2 ITRs, we observed only a limited number of recombination sites and concluded that Ad2 ITRs play a role in illegitimate recombination with the AAV-ITRs that leads to generation of biologically active wild type-like AAV. Consequently, by removing the Ad2 ITRs from the helper plasmid, nearly 5-fold reduction in the illegitimate recombination frequency can be achieved.
[0056] The first 10 nucleotides in the D-sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome. See, Wang,
[0057] Four recombinant AAV vectors, pD-5, pD-10, pD-15 and pD-20, were constructed as follows. Plasmid pXS-22 can be employed as starting material. The plasmid pXS-22 can be obtained from a public depository or constructed following the methods described in Wang,
D-sequence: 5′ CTCCA TCACT AGGGG TTCCT 3′ GAGGT AGTGA TCCCC AAGGA 5′ S-sequence: 5′ CCAA TATTA GATCT GATAT CA 3′ 3′ GGTT ATAAT CTAGA CTATA GTGAT C 5′
[0058] Four additional oligonucleotide sequences were synthesized which contained selected nucleotides identical to the authentic or native D-sequence in place of nucleotides in the S-sequence. These four oligonucleotides are:
D-5 oligonucleotide: 5′ CCAA 3′ GGTT D-10 oligonucleotide: 5′ CCAA 3′ GGTT D-15 oligonucleotide: 5′ CCAA 3′ GGTT D-20 oligonucleotide: 5′ CCAA 3′ GGTT
[0059] The selected nucleotides conforming to the authentic, native, D-sequence in the AAV ITR are indicated above in bold.
[0060] The D-5, D-10, D-15 and D-20 oligonucleotide sequences were each inserted between the Xba I and Bal I sites of plasmid pXS-22, which is described in Wang,
[0061] Each of the four foregoing recombinant AAV vectors, pD-5, pD-10, pD-15 and pD-20 may be employed in the methods of the invention. We have determined that to optimize packaging 10 of the native D-nucleotides are sufficient. The most preferred native 10 D-nucleotides are those included in the pD-10 vector and indicated in bold in the D-10 oligonucleotide sequence above. The pD-15 and pD-20 vectors, or their respective indicated oligonucleotides (see above), may be used but they contain extra, unnecessary nucleotides that would advantageously be eliminated in order to allow for more space in the AAV vector for nucleotides encoding the desired therapeutic molecule. The pD-5 vector works, but with less efficiency. Consequently, the absolute minimal necessary sequence is the 5 nucleotide sequence enumerated in bold in the D-5 oligonucleotide sequence above and contained in the pD-5 vector. The pD-10 vector allows for the insertion of an additional 106 nucleotides.
[0062] Nucleic acid sequences encoding therapeutic molecules can be ligated between the ITRs of these vectors using known techniques. The vectors or virions may be formulated into pharmaceutical compositions for administration in human or other mammalian patients.
[0063] Plasmid pXS-22 was deposited on Sep. 10, 1996 with the ATCC, 12301 Parklawn Drive, Rockville, Md., USA under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. The Accession Number is 97710. This deposit assures maintenance of a viable culture for 30 years from the date of deposit. The organism(s) deposited will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between applicant and the ATCC that assures unrestricted availability upon issuance of the pertinent U.S. patent. This deposit is provided as convenience to those of skill in the art, and is not an admission that a deposit is required under 35 U.S.C. 112. The nucleic acid sequence of this deposit, as well as the amino acid sequence of the polypeptide(s) encoded thereby, are incorporated herein by reference and should be referred to in the event of an error in the sequence described herein. A license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.
[0064] All patents, patent publications, patent applications and scientific articles mentioned in this specification are herein incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.