MXPA99002215A - Method for recombinant adeno-associated virus-directed gene therapy - Google Patents

Abstract

A method of prolonging gene expression by reducing immune response to a recombinant adeno-associated virus (AAV) bearing a desired gene administered into the muscle of a mammal is described.

Description

METHOD FOR GENE THERAPY DIRECTED THROUGH ADENOSIS RECOMBINANT VIRUS This work was supported by the National Institutes of Health, Concession No. DK47757. The government of E.U.A. You have certain rights to this invention.

BACKGROUND OF THE INVENTION The adeno-associated virus (AAV) is a replication-defective parvovirus whose genome is approximately 4.6 kb in length, and includes inverted terminal repeats (ITRs) of 145 nucleotides. The AAV single-stranded DNA genome contains genes responsible for replication (rep) and virion formation (cap). When this non-human pathogenic virus infects a human cell, the viral genome is integrated into chromosome 19, resulting in the latent infection of the cell. Infectious virus production and virus replication do not occur unless the cell is coinfected with a lytic helper virus such as an adenovirus or herpes virus. After infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced.

The AAV has unique characteristics that make it attractive as a vector to release DNA introduced into cells. Several groups have studied the potential use of AAV in the treatment of disease states. Studies of recombinant AAV (rAAV) in vitro have been in disagreement due to low transduction frequencies; incubation of cells with rAAV in the absence of contaminating wild-type AAV or helper adenovirus is associated with poor expression of the recombinant gene [D. Russell and others, Proc. Nati Acad. Sci. U.S.A., 91: 8915-8919 (1994); I. Alexander and others, J. Virol. , 68 .: 8282-8287 (1994); D. Russell and others, Proc. Nati Acad. Sci. U.S.A., 92: 5719-5723 (1995); K. Fisher et al., J. Virol. , 70.:520-532 (1996); and F. Ferrari et al., J. Virol. , 70: 3227-3234 (1996)]. In addition, the integration is inefficient and not directed towards chromosome 19 when rep is absent [S. Kumar et al., J. Mol. Biol. , 222 .: 45-57 (1991)]. AAV transduction is substantially increased in the presence of adenovirus because the single chain rAAV genome is converted to an unintegrated double stranded intermediate, which is transcriptionally active [K. Fisher et al., J. Virol. , 20: 520-532 (1996); and F. Ferrari et al., J. Virol. , 70: 3227-3234 (1996)]. The adenovirus increases the transduction of rAAV by expressing the product of the early E4 ORF6 gene [K. Fisher et al., J. Virol. , 70.:520-532 (1996); and F. Ferrari et al., J. Virol., 70: 3227-3234 (1996)].

The performance of rAAV has been combined as a vector for in vivo models of gene therapy. The most promising results have been in the central nervous system, where stable transduction has been achieved in postmitotic cells [M. Kaplitt et al., Nat. Genet , .8: 148-154 (1994)]. Incubation of bone marrow cells ex vivo with rAAV produces some transduction, although a stable and efficient hematopoietic graft has not been demonstrated in transplant models [J. Miller et al., Proc. Nati Acad. Sci. U.S.A., 91: 10183-10187 (1994); G. Podsakoff et al., J. Virol. , 68: 5656-5666 (1994); and C. Walsh et al., J. Clin. Invest., 94: 1440-1448 (1994)]. Administration of rAAV in the airway or blood results in gene transfer into lung epithelial cells [K. Fisher et al., J. Virol. , 70: 520-532 (1996); and T. Flotte et al., Proc. Nati Acad. Sci. U.S.A., 90 .: 10613-10617 (1993)] and hepatocytes [K. Fisher et al., J. Virol. , 70.:520-532 (1996)], respectively; however, transgenic expression has been found to be low, unless the adenovirus is present [K. Fisher et al., J. Virol. , 70.:520-532 (1996)]. What is needed is a method to improve gene transfer mediated by rAAV.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for improving the expression of a selected gene released to an animal by recombinant AAV. The method involves introducing a recombinant AW vector comprising a desired transgene into a muscle cell in the absence of a helper virus. The vector can be administered in cardiac muscle, smooth, or preferably, skeletal. In a preferred embodiment, the transgene released by rAAV codes for a secretable and / or diffusible product which is therapeutically useful. In this embodiment, the transgenic product may have a therapeutic effect on sites remote from the site of release. In another embodiment, the transgene codes for a non-secretable product (e.g., a dystrophin polypeptide), which is desired to be released into the muscle (e.g., for the treatment of muscular dystrophy). In another aspect, the present invention provides a method for treating an animal suffering from hemophilia. The method involves administering to the animal muscle a recombinant adeno-associated virus comprising the gene encoding factor IX, and sequences that regulate gene expression. In still another aspect, the present invention provides a method for treating an animal with atherosclerosis. The method involves administering in the animal muscle a recombinant adeno-associated virus comprising the gene encoding ApoE, and regulatory sequences capable of expressing said gene. Other aspects and advantages of the present invention are further described in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration showing the linear arrangement of AAV.CMVLacZ (4883 bp). Relevant elements include AAV ITRs (compact black boxes), CMV promoter (shaded arrows), SV40 intron and polyadenylation (clear boxes), and lacZ DNA (shaded box). The location of a cDNA probe that can be used to detect the internal BamHI fragment, as well as the full length vector is also shown. Figure 2A is a schematic illustration of the linear arrangement of the AAV.CMVLazcZ concatamer. Relevant features include AAV ITRs (shaded boxes), CMV enhancer / promoter (solid black arrow), SV40 intron and polyadenylation (light squares) and LacZ cDNA (shaded box). The monomer of AAV.CMVLacZ is shown bound in accordance with an end-to-end direct ligation mechanism (marked as J) in the ITRs. Therefore, two copies of the AAV ITRs in the union are present in the scheme. Figure 2B is a schematic illustration showing an amplified view of the binding domain. The relevant characteristics are as indicated in Figure 3A above. The horizontal arrows indicate the location and direction of the PCR primers used to amplify through the binding of proviruses. Initiator 005 is a sense chain starter. The primers 013 and 017 are antisense strand initiators. Figure 3 is a schematic illustration showing the predicted PCR product involving direct double-sided end-to-end ligation of monomeric genomes of AAV.CMVLacZ. Two complete ITRs (shaded boxes) with their respective "FLOP" and "FLIP" orientation are shown at the junction (marked as J). The CMV promoter (compact black box) and the polyadenylation signal (light box) are also indicated. The PCR cloning site in pCRII is flanked by EcoRI sites, as shown. The location of 3 SnaBI sites located within the PCR product is also shown. Initiators 005 and 013 are also indicated. Figure 4A, in conjunction with Figures 4B to 4G, illustrate the structure of PCR products that map to the head-to-tail junction of the AAV.CMVLasZ concatamers of Figure 4A. Figure 4A shows the predicted PCR product assuming a direct double end-to-end ligation of monomeric genomes of AAV.CMVLacZ. They are shown in the union (marked as j) two complete ITRs (shaded box), with their respective orientation of "FLOP" and "FLIP". The CMV promoter (compact black box) and the polyadenylation signal (light box) are also indicated. Figure 4B illustrates the structure of the PCR product of clone 3. Figure 4C illustrates the structure of the PCR product of clone 8. Clone 8 is almost identical in size to clone 3, but contains a different arrangement than ITR union. Figure 4D illustrates the structure of the PCR product of clone 5. Figure 4E illustrates the structure of the PCR product of clone 2. Figure 4F illustrates the structure of the PCR product of clone 6. Figure 4G illustrates the structure of the PCR product of clone 7. Figure 5A characterizes lymphocyte activation Cytotoxic T directed against the adenoviral antigen, as well as lacZ. This is an analysis of harvested lymphocytes from group 1 of example 5. Figure 5B characterizes the activation of cytotoxic T lymphocytes directed against the adenoviral antigen, as well as lacZ. This is an analysis of harvested lymphocytes from group 2 of example 5. Figure 5C characterizes the activation of cytotoxic T lymphocytes directed against the adenoviral antigen, as well as lacZ. This is an analysis of harvested lymphocytes from group 3 of example 5. Figure 6A shows the activation of T lymphocytes in response to different antigens including AAV purified for β-galactosidase, or adenovirus, for each of groups 1 to 4 of Example 5. The activation is demonstrated by the secretion of IFN-gamma representing the TH1 subset of T cells. Figure 6B shows the activation of T lymphocytes in response to different antigens including AAV purified for β-galactosidase, or adenovirus, for each of groups 1 to 4 of Example 5. The activation is demonstrated by the secretion of IL-10 representing the TH2 subset of T cells. Figure 7A illustrates the results of an enzyme-linked immunosorbent assay (ELISA), which shows the development of antibodies directed against β-galactosidase in various groups of example 5. Figure 7B illustrates the results of an enzyme-linked immunosorbent assay (ELISA), showing the development of antibodies directed against adenovirus type 5 in several groups of example 5.

Figure 8 is a graph of plasma hF.IX concentration in C57BL / 6 mice as a function of time after IM injection of 2x1o11 rAAV-hF.IX / animal vector genomes (n = 4). Figure 9 is a graph showing the circulating antibody against human F.IX as a result of intramuscular injection of rAAV-hF.IX in C57BL / 6 mice. The time course of anti-hF.IX antibody concentration in plasma after injection with 2x10 vector / animal genomes (n = 3), was determined by ELISA test using anti-hF.IX Mouse Mab [Boehringer Mannheim] as standard. Each line represents an individual animal. Figure 10 illustrates the concentration of hF.IX in plasma in three mice as a function of time after injection. Each symbol represents a different animal. The fourth animal in this experiment died 5 weeks after the injection after traumatic phlebotomy. Figure 11 is a graph illustrating the concentration of hF. IX in plasma in four Rag-1 mice as a function of time after injection with rAAV-hF.IX.

Each symbol represents a different animal. Figure 12 is a schematic diagram of a head-to-tail concator of rAAV present in transduced cells. Figure 13 is a schematic diagram illustrating the construction of A. CMVApoE.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for muscle-directed adenovirus-mediated gene transfer (AAV), which provides high-level and stable expression of a transgene in the absence of helper viruses or exogenous helper molecules. Particularly, this method consists in introducing a recombinant AAV that possesses a desired transgene in a muscle cell. Conveniently, the rAAV vector is injected directly into cardiac, skeletal, or smooth muscle, and the transgene codes for a secreted and / or diffusible therapeutic product, such as a polypeptide or an RNA molecule. However, the method of the invention is equally useful for the administration of nucleic acids encoding therapeutically useful non-secreted products. Particularly, the inventors have found that intramuscular injection of purified rAAV free of auxiliary molecules (i.e., rAAV that is substantially free of contamination with adenovirus or wild-type AAV), produces a highly efficient transduction of muscle fibers, leading to the stable and prolonged expression of the transgene. By free of auxiliary molecules it is further understood that the AAV is in the substantial absence of helper viruses or other exogenous helper molecules (ie, helper molecules not native to, or normally present in, muscle cells). This is achieved without significant inflammation or activation of immune responses to the transgenic product, despite the fact that the product may be a new antigen. The stability of the transgenic expression produced in accordance with the methods of this invention is particularly impressive. While not wishing to be limited by theory, it is thought that this stability is due to the highly inefficient chromosomal integration of the proviral DNA of AAV into muscle cells in the absence of helper viruses or other exogenous helper molecules. Several observations support this hypothesis. As described in the following examples, analysis of Hirt extracts did not allow to detect a double-stranded episomal form of the viral genome. Southern analysis of total cellular DNA revealed a defined band when it was digested with an enzyme that has two cleavage sites within the AAV vector, whereas no defined band was observed when the same DNA was digested with an enzyme that has no sites within the viral genome. Additional DNA analysis focused on the formation of concatamers and their structure. Previous studies of lytic AAV infections have shown that episomal replication of rAAV proceeds through head-to-head or tail-to-tail concatamers, while latent infections that result in the integration of the provirus are established as dual provisions of head with tail [KI Berns, Microbiol. Rev., 54. = 316-329 (1990); J. -D. Tratschin et al., Mol. Cell. Biol., 5: 3251-3260 (1985); N. Muzcyzska, Current Topics in Microbiology and Immunoloqy, 158: 97-129 (1993); S.K. McLauglin et al., J. Virol., 62: 1963-1973 (1988)]. The data given herein demonstrate that the muscle cells transduced with rAAV vectors according to this invention contain concatamers consisting of double head-to-tail arrangements with variable deletions of both ITRs, consistent with a transduction mechanism that involves the integration of the rAAV provirus. Sequence analysis of the junctions created by rAAV concatamers indicated consistent but variable suppressions of both ITRs. Fluorescent analysis of in situ hybridization (FISH) was consistent with individual integration sites in approximately 1 in 20 nuclei, whereas Southern analysis indicated an average of 1 proviral genome per diploid muscle fiber genome. Taken together, these findings indicate that the average concatomeres comprise at least 10 proviral genomes. Another advantage of the method of the invention is the surprising absence of inflammation after administration of therapeutic doses of the vector. For example, C57BL / 6 mice injected with an AAV vector for lacZ could not produce a humoral immune response to E. coli beta-galactosidase despite the fact that anti-beta-galactosidase vibrating antibodies were produced in these animals when Injected an adenoviral vector for lacZ in the skeletal muscle. Thus, when the AAV vector free of auxiliary molecules was used in accordance with the method of this invention, immune responses to the transgene were modulated. This contrasts markedly with prior art methods of gene transfer, such as those using naked plasmid DNA [J. A. Wolf et al., Science, 247: 1465-1468 (1990) or adenovirus-mediated gene therapy [S.K. Tripathy et al., Nat. Med., 2: 545-550 (1996), which typically induces strong immune responses to the transgene. Thus, the method of the invention provides a significant advantage over other gene delivery systems, particularly with respect to the treatment of chronic disorders that may require repeated administrations.

I. The recombinant AAV A recombinant AAV vector possessing a selected transgene is used in the methods of the invention. In addition to the transgene, the vector also contains regulatory sequences that control the expression of the transgene in a host cell, for example, a muscle cell. Many rAAV vectors are known to those skilled in the art, and the invention is not limited to any particular rAAV vector. For example, suitable AAV vectors, and methods for producing them, are described in the U.S. patent. No. 5,252,479; patent of E.U.A. Do not. ,139,941; International Patent Application No. W094 / 13788; and International Patent Application No. W093 / 24641. A particularly suitable vector is described below.

A. AAV sequences Currently, a preferred rAAV is deleted from all viral open reading frames (ORFs), and retains only the 5 'and 3' inverted terminal repeat (ITRs) sequences that act at the cis-position [see, for example, BJ Carter, in "Handbook of Parvoviruses", ed. , P. Tijsser, CRC Press, pp. 155-168 (1990)]. Thus, the sequences encoding the rep and cap polypeptide are deleted. The ATR ITR sequences are approximately 143 bp in length. Although it is preferred that substantially the entire 5 'and 3' sequences comprising the ITRs are used in the vectors, one skilled in the art will understand that some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences, while retaining their biological functions, is within the scope of the technique. See, for example, texts such as Sambrook et al., "Molecular Cloning, A Laboratory Manual." 2Z ed. , Cold Spring Harbor Laboratory, New York (1989). AAV ITR sequences can be obtained from any known AAV, including the types of human AAV currently identified. It is anticipated that the selection of the AAV type does not limit the invention. Various types of AAV, including types 1 to 4, are available from the American Type Culture Collection, or available by request from various commercial and institutional sources. Similarly, AAVs that are known to infect other animals can also be employed in the vector used in the methods of this invention. In the examples given herein, an AAV-2 is used for convenience. Specifically, the 5 'and 3' AAV ITR sequences of AAV-2 flank a selected transgenic sequence and associated regulatory elements, as described below.

B. The transqen The transgene sequence contained within the rAAV vector is a nucleic acid sequence, heterologous to the AAV sequence, which codes for an RNA or polypeptide of interest. The transgene is operatively linked to regulatory components, so as to allow expression of the transgene in muscle cells. The composition of the transgene sequence will depend on the use of the resulting vector. For example, a type of transgene sequence includes the sequence of a reporter gene, which after expression produces a detectable seAal. Said reporter gene sequence includes, without limitation, an AD? C for E. coli beta-galactosidase (LacZ), a gene for alkaline phosphatase and a gene for crude fluorescent protein. These sequences, when associated with regulatory elements that drive their expression, provide detectable signals by conventional means, for example, ultraviolet light wavelength absorbance, visible color change, etc. The expression of said transgenes can be used in methods for cell quantification, cell identification, and the like. A more preferred transgenic sequence includes a therapeutic gene that codes for a desired gene product. These therapeutic nucleic acid sequences typically encode for products that, when administered to a patient in vivo or ex vivo, are able to replace or correct an inherited or non-inherited genetic defect, or treat an epigenetic disorder or disease. The method of the invention, which allows the transgene to be released into muscle cells, is particularly well suited for use in connection with secreted therapeutic proteins, such as factor IX, useful in the treatment of hemophilia, or apolipoprotein (Apo) E , useful in the treatment of atherosclerosis. However, other therapeutic gene products, particularly those that are secreted, can be easily selected by the person skilled in the art. Examples of genes encoding secreted and / or diffusible products include, without limitation, cytokines, growth factors, hormones, differentiating factors and the like, for example, beta-interferon (beta-IFN), erythropoietin (epo), insulin, Growth hormone (GH) and parathyroid hormone (PTH). These genes are useful for the treatment of several conditions, including multiple sclerosis and cancer (beta-IFN), anemia (epo), diabetes (insulin), small stature (GH) and osteoporosis (PTH). The method of the invention is also useful for releasing genes encoding products not secreted into the muscle. For example, it is anticipated that the method of the invention is useful in the treatment of muscular dystrophies, since it allows the release of a gene for diastrophin [see, for example, C.C. Lee et al., Nature, 349: 334-336 (1991)] by a rAAV in accordance with the method of the invention. The selection of the transgene is not considered to be a limitation of this invention, since said selection is within the knowledge of the person skilled in the art.

C. Vector regulatory elements In addition to the transgene and ITR sequences of the AAV, the vector also includes regulatory elements necessary to direct the expression of the transgene in transduced muscle cells. Thus, the vector conveniently contains a selected and enhancer promoter (if desired), which are operably linked to the transgene and located therein, between the vector AAV ITR sequences.

The selection of the promoter and, if desired, of the enhancer is a routine aspect and is not a limitation of the vector itself. Useful promoters can be constitutive promoters or regulated (inducible) promoters, which will allow the controlled expression of the transgene. For example, a convenient promoter is that of the cytomegalovirus immediate early enhancer / promoter [see, for example, Boshart et al., Cell, 41: 521-530 (1985)]. Other suitable promoters include, without limitation, the LTR enhancer / promoter of Rous sarcoma virus and the mouse metallothionin inducible promoter. Other promoter / enhancer sequences may be selected by the person skilled in the art. The vectors will also conveniently contain nucleic acid sequences that affect the transcription or translation of the transgene, including sequences that provide signals required for efficient polyadenylation of the transcript, and introns with functional splice donor and acceptor sites. A common poly-A sequence that is used in the vector examples of this invention is that derived from SV-40 of papovavirus. The poly-A sequence is generally inserted into the vector after the transgene sequences and before the 3 'AAV ITR sequence. A common intron sequence is also derived from SV-40, and is referred to as the intron sequence T of SV-40. The selection of these and other desirable elements to control or increase gene expression are conventional, and many such sequences are known to those skilled in the art [see, for example, Sambrook et al., And references cited therein]. The combination of the transgene, promoter / enhancer and the other regulatory elements, is referred to herein as a "minigene" to facilitate its reference herein. As noted above, the minigene is flanked by the 5 'and 3' AAV ITR sequences. Provided with the teachings of this invention, the design of said minigen can easily be achieved by the person skilled in the art. An example of a rAAV, that is, AAV.CMVLacZ, and its use in the method of the invention, is provided in the following examples. As illustrated in Figure 1, this example of rAAV contains a 5 'AAV ITR, a CMV promoter, an SV-40 intron, a transgene for LacZ, a poly-A sequence of SV-40 and an ITR of AAV 3 '. However, as noted above, the method of this invention is not limited to the use of any particular rAAV.

D. Production of rAAV The sequences used for the construction of the rAAV used in the method of this invention can be obtained from commercial or academic sources based on previously published and described materials. These materials can also be obtained from an individual patient, or they can be generated and selected using standard recombinant molecular cloning techniques known and practiced by those skilled in the art. Any modification of the existing nucleic acid sequences used in the production of the rAAV vectors, including deletions, insertions and other mutations in the sequences, can also be generated using standard techniques. The rAAV assembly, including the AAV sequences, the transgene and other elements of the vector, can be achieved using conventional techniques. A particularly convenient technique is described in K. J. Fisher et al., J. Virol., 70 (1): 520-532 (January 1996), incorporated herein by reference. Nevertheless, other suitable techniques include cloning of cDNAs, such as those described in texts [Sambrook et al., cited above], the use of overlapping oligonucleotide sequences of the AAV genome, the polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. When appropriate, standard transfection and cotransfection techniques are used to propagate the rAAVs in the presence of helper viruses, for example, adenoviruses deleted for El using techniques such as CaP? 4 transfection techniques, and can be easily selected by the skilled artisan. The technique. Other conventional methods that can be used in this invention include homologous recombination of AW viral genomes, virus plating on agar layer, methods for measuring seAn generation, and the like. Conveniently, the rAAV produced is purified to remove any adenovirus or wild-type contaminating AW. A particularly convenient purification scheme is described in K. J. Fisher et al., J. Virol. , 70 1): 520-532 (January 1996), incorporated herein by reference. However, one skilled in the art can easily select other suitable purification means.

II. Therapeutic Applications Once a rAAV containing a desired transgene is obtained, the vector is administered directly into the muscle of an animal. An advantage of the method of the invention is that the muscle is particularly well suited as a site for the production of secreted therapeutic products, such as factor IX or apolipoprotein (Apo) E, among others. Alternatively, the method of the invention is used to deliver a non-secreted gene product to muscle cells. The rAAV vectors of the present invention can be administered to a patient, preferably suspended in a biologically compatible solution, pharmaceutically acceptable carrier or delivery vehicle. A suitable vehicle includes sterile saline. Other sterile aqueous and non-aqueous isotonic injectable solutions and sterile aqueous and non-aqueous suspensions which are known to be pharmaceutically acceptable carriers and well known to those skilled in the art can be used for this purpose. The rAAV vectors of this invention are administered in sufficient amounts to provide for the integration and expression of the selected transgene, so that a therapeutic benefit can be obtained without undue adverse effects and with medically acceptable physiological effects that can be determined by science experts. medical In a preferred embodiment, the rAAV is injected directly into the cardiac, skeletal, or smooth muscle. The person skilled in the art will also appreciate that other methods of administration, for example, intravenous or in rarterial injection, can also be used in the method of the invention as long as rAAV is directed towards the muscle cells. The dosage of the rAAV vector will depend mainly on factors such as the condition being treated, the selected transgene, and the age, weight and health of the patient, and may thus vary between patients. It is thought that a therapeutically effective dose of the rAAV of the present invention is on the scale of about 1 to about 50 ml of saline containing concentrations of about 1 x 10 ° to 1 x 10 particles / ml rAAV vector of the present invention. Conveniently, each dose contains at least 10 particles of rAAV. A most preferred dosage for human is about 1 to 20 ml of saline at the above concentrations. The expression levels of the selected transgene can be monitored by bioassay to determine the route, dose or frequency of administration. The administration of rAAV can be repeated, as necessary. The examples given below illustrate the preferred methods for preparing the vectors, and for carrying out the methods of the invention. These examples are illustrative only, and do not limit the scope of the invention.

EXAMPLE 1 Production of AAV.CMVLacZ A recombinant AW (rAAV) was generated in which the rep and cap genes were replaced with a minigene expressing E. coli β-galactosidase under the control of a CMV promoter (AAV.CMVlacZ). AAV.CMVlacZ was produced by the use of the pAAV plasmid. CMVlacZ, which acts at the level of the cis position, which was derived from psub201 [R. Samulski et al., J. Virol. , 61 (10): 3096-3101 (1987). For a short time, the plasmid was transfected into 293 cells with adenovirus deleted in El [K. J. Fisher et al., J. Virol. , 70: 520-532 (1996)], and the rep and cap functions were provided by a transactant plasmid, pAAV / Ad [R. Samulski et al., J. Virol. , 63: 3822-3826 (1989)]. The production batches of the AAV.CMVLacZV vector were titrated in accordance with the genome / ml copies, as described [Fisher et al., J. Virol. , 70: 520-532 (1996)]. The 5 'to 3' organization of the AAV.CMVLacZ genome (4883 bp), includes: the 5 'AAV ITR (pb 1-173) obtained by PCR using pAV2 [C. A. Laughlin and others. Gene, 23: 65-73 (1983)] as a template [nucleotide numbers 365-538 of SEQ ID NO: 1], an immediate CMV enhancer / early promoter [Boshart et al., Cell, 41: 521-530 (1985 ); nucleotide numbers 563-1157 of SEQ ID NO: 1], an SV40 intron (numbers of nucleotides 1178-1179 of SEQ ID NO: 1), a lacZ cDNA of E. coli (numbers of nucleotides 1356-4827 of SEQ ID NO: 1), an SV40 polyadenylation signal (a restriction fragment 237 Bam HI-BclI containing the cut-off signals / poly-A of the early and late transcription units, nucleotide numbers 4839-5037 of SEQ ID NO : 1), and the AW 3 'ITR, obtained from pAV2 as a SnaBI-BglII fragment (nucleotide numbers 5053-5221 of SEQ ID NO: 1) .Figure 1, two Bam Hl sites are present in the double-stranded vector sequence.The first is located in the SV40 intron at the 875 bp position, and the second is located between the lacZ cDNA and the SV40 polyadenylation signal at the 4469 bp position. , the digestion of the double chain sequence with BamHl releases a 3595 bp fragment in length. of the cDNA probe that can be used to detect the internal BamHI fragment, as well as the full length vector. The rAAV.CMVlacZ virus was purified using standard techniques [see, for example, K. F. Kozarsky et al., J. Biol. Chem., 269: 13695-13702 (1994)]. RAAV supply materials used in the following examples were tested to ensure the absence of replication-competent wild-type AAV and helper adenoviruses deleted in El, as follows: 293 cells were seeded on camera slides and coinfected with adenovirus wild type and an aliquot of the rAAV vector supply material. Twenty-four hours after infection, the cells were fixed and incubated with a mouse monoclonal antibody against AAV capsid proteins (American Research Products). The antigen-antibody complex was detected with a secondary antibody conjugated with FITC. A positive seAa.1 was considered as an infectious AAV unit. The contaminating auxiliary adenovirus was evaluated by infecting 293 cells with an aliquot of the rAAV vector delivery material, and typing for reporter gene expression for alkaline phosphatase. The helper adenovirus is deleted in El and contains a cDNA for human placental alkaline phosphatase under the transcriptional control of the CMV promoter. No helper adenoviruses or competent replication wild-type AAV were detected in the highly purified rAAV preparations.

EXAMPLE 2 rAAV stably translates skeletal muscle in vivo RAAV was administered with and without adenovirus deleted in E2a, which was used for the purpose of increasing transduction. Procedures in animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wistar Institute. For a short time, five-week-old female C57BL / 6 mice (Jackson Laboratories, Bar Harbor, Maine) were anesthetized with an intraperitoneal injection of ketamine (70 mg / kg) and xylazine (10 mg / kg), and a Subsequent incision of 1 cm in its lower extremity. Samples of rAAV.CMVLacZ (1 x 109 vector genomes) in 25 SI of saline regulated at its pH with HEPES (HBS, pH 7.8) or rAAV. CMVlacZ complemented with an adenovirus mutant E2a, dl802 [S. A. Rice and D. L. Klessig, J. Virol. , 56: 767-778 (1985)] (5x10 particles of? -260 '1 10 pfu) shortly before injection, were injected into the tibialis anterior muscle of each paw using a Hamilton syringe. The incisions were closed with Vicryl 4-0 suture. To analyze the expression of the transgene, the animals were necropsied at various time points after the injection, and the injected muscle was excised with a scalpel. The tissue was placed on a drop of the OTC inclusion compound, quickly frozen in isopentane cooled with liquid nitrogen for seven seconds, and immediately transferred to liquid nitrogen. Tissue analysis at each time point represented a minimum of 6 injection sites (ie, bilateral sampling of at least 3 animals). For histochemical analysis, the frozen muscle was laterally segmented into two equal halves, generating a cross-sectional face of the tissue. Both halves of the tissue were sectioned in series (6 Sm). For X-gal histochemistry, the sections were fixed in a freshly prepared 0.5% glutaraldehyde solution in PBS, and stained for β-galactosidase activity, as described [K. J. Fisher et al., J. Virol. , 70: 520-532 (1996)]. The sections were contracted in neutral red solution, and assembled. When the adenovirus was used as an adjunct to rAAV, the histochemical analysis of X-gal revealed high level transduction in muscle fibers around day 17 associated with substantial inflammation. Surprisingly, however, animals that received rAAV in the absence of auxiliary adenovirus demonstrated transduction levels that exceeded those found in the presence of adenovirus. These high levels have persisted without apparent decrease for 180 days.

EXAMPLE 3 The rAAV qenoma is integrated with high efficiency as truncated concatamers head to tail To characterize the molecular state of the stabilized genome of rAAV, Southern blot analysis of skeletal muscle DNA harvested from injected mice was carried out as described above. We considered models in which the rAAV genome persists as a double-stranded episomal genome, such as that formed during lytic infection, or as an integrated provirus that resembles latent infection. For a short time, low molecular weight DNA (Hirt) (see Part A below) and high molecular weight genomic DNA (see Part B below), were isolated from the mouse muscle at selected time points. The DNA samples were resolved on a 1% agarose gel, and electrophoretically transferred to a nylon membrane (Hybond-N, Amersham). The blot was hybridized with a 32 P-labeled restriction fragment of random primer dCTP isolated from the lacZ cDNA.

A. Detection of the double-stranded episomal genome To detect the non-integrated forms of the rAAV genome, Hirt extracts of the transduced muscle DNA were analyzed by hybridization with a P-labeled cDNA that maps to the probe sequence shown in Figure 1 The samples of Hirt DNA (15 Si, equivalent to 15 mg of tissue) were extracted from the harvested muscle on days 8, 17, 30 and 64 after the injection. DNA was analyzed from a line of cultured cells infected with rAAV in the presence of adenovirus. The analysis of Hirt extracts from that cell line demonstrated the presence of monomeric forms of the virus both single chain and double chain. However, Hirt extracts of muscle transduced with rAAV alone demonstrated a single-chain genome around day 8, which decreased to undetectable levels around day 64. No forms of double-chain rAAV were detected in Hirt extracts, even when the filters were overexposed. This indicates that the single chain rAAV genome is efficiently transferred into skeletal muscle cells; however, it is not converted to transcriptionally active episomal forms.

B. Detection and characterization of integrated proviral DNA To detect integrated proviral DNA, additional hybridization studies were carried out with total cell DNA harvested from skeletal muscle transduced 64 days after infection. Genomic DNA (10 Sg, equivalent to 18 Sg of tissue) was digested with BamHl or HindIII, a restriction enzyme that does not cut proviral DNA. As expected, digestion with HindIII resulted in a stain after fractionation of the gel, and hybridization with a virus-specific probe. However, when the genomic DNA was digested with BamHl, which cuts twice within the provirus, a defined band of the predicted size of 3.6 kb was detected at an abundance of about 1 proviral genome / diploid host cell genome. The structure of the integrated provirus was characterized using PCR analysis to delineate the potential mechanisms of persistence. Previous studies of the wild type and rAAV have suggested different DNA replication pathways in the lytic and latent phases of the viral life cycle. Specifically, in the presence of helper virus, the AAV replicates to form dimeric replicative intermediates by a mechanism that results in the synthesis of head-to-head or tail-to-tail concatamers. This contrasts with latent infections, where the integrated proviral genome is characterized by genomic head-to-tail arrangements. Skeletal muscle genomic DNA was subjected to PCR analysis to amplify the junctions between AAV genomic concatemers. A PCR method was developed to detect the integrated rAAV, based on data indicating that the integrated forms of rAAV are typically found as head-to-tail concatemers. Specifically, oligonucleotide primers were synthesized to allow selective PCR amplification through head-to-tail junctions of 2 monomers of the AAV.CMVLacZ genome. The sense chain primer 005 (5 '-ATAAGCTGCAATAAACAAGT-3 *; SEQ ID NO: 4) mapped to the 4584-4603 bp position of the SV40 polyadenylation signal domain. The antisense chain primer 013 (5 »-CATGGTAATAGCGATGACTA-3 •; SEQ ID NO: 2) mapped to the 497-478 bp position of the CMV promoter, while the 017 antisense chain primer (S'-GCTCTGCTTATATAGACCTC-S) ', * SEQ ID NO: 3) mapped to the 700-680 bp position of the CMV promoter. If the ITRs are retained intact, oligomers 005 + 013 [SEQ ID NO: 2] must amplify a 797 bp fragment, while oligomers 005 and 017 [SEQ ID NO: 3] must amplify a 1000 bp fragment. It is important to emphasize that the predicted sizes of the PCR product are based on the assumption that the junctions of the provirus contain two copies of ITR. The amplification through a binding having less than two copies, will therefore generate a PCR product that is proportionally smaller in size. PCR reactions were carried out using 100 ng of genomic DNA template and initiator concentrations of 0.5 SM. The profile of the thermocycle was 94 ° C 1 min, 52 ° C 1 min and 72 ° C 1 min 30 sec for 35 cycles; and the denaturation step at 94 ° C of the first cycle was 2 minutes, while the extension step at 72 ° C of the last cycle was 10 minutes. The PCR products were analyzed by agarose gel electrophoresis. PCR reactions were carried out on genomic DNA isolated from muscle transduced by AAV.CMVLacZ harvested on day 64 after infection, as described above. As a negative PCR control, muscle genomic DNA injected with saline solution regulated in its pH by Hepes (HBS) was used. No amplification products were detected when primers were used that must encompass a head-to-head or tail-to-tail joint (data not shown). However, when the muscle DNA transduced by AAV.CMVlacZ was analyzed with oligonucleotides 005 and 017, a stain consistent with a heterogeneous population of head to tail concatamers was detected (Figs 2A and 2B). PCR reactions were also carried out with genomic DNA from cell lines containing integrated AAV.CMVLacZ. The provirus structure of these clones has been determined by Southern blot analysis. Three cell lines were identified (10-3.AV5, 10-3.AV6 and 10-3.AV18), each of which contains at least two copies of integrated AAV.CMVLacZ monomers arranged head to tail. Based on the size of the PCR products (a product of 720 bp using the primer series 005-013, and a product of 930 bp using the primer series 005-017), two clones, 10-3.AV5 and 10-3. V6, probably contain 1.5 copies of the ATR ITR in the junction. Another clone, 10-3.AV18, contains a large deletion spanning the AAV ITRs generating a 320 bp product using the primer series 005-013, and a 500 bp product using the primer series 005-017. Another cell line, 10-3.AV9, contains a single monomer copy of AAV.CMVLacZ integrated in accordance with Southern blotting, and appears to be confirmed by the presence of a PCR product. Thus, DNA analysis of rAAV-infected cell lines, selected for stable transduction, revealed distinct bands smaller than those predicted for an intact head-to-tail concatamer.

D. Structural analysis Detailed structural analysis of the proviral junctions recovered from the skeletal muscle DNA was carried out by cloning from the PCR reaction (Figure 3), followed by restriction analysis (Figures 4A-4G). Particularly, the PCR products of one of the muscle samples, BL.ll, obtained as described above, were ligated directly into the commercially available pCRII plasmid, in which the insert was flanked by EcoRI sites. The commercially available TOP10 F 'competent bacterial strain was transformed by ligation reactions. In effect, this procedure results in a library of PCR products of the plasmid. The library was plated at a certain density to give well-isolated colonies, and was selected by coating with a nylon membrane, and hybridizing with a 32 P-labeled fragment corresponding to the CMV promoter / enhancer. Putative positive clones were grown overnight in small-scale cultures (2 ml). Plasmid DNA was extracted from 6 representative clones of the cultures at small scale, and was digested with EcoRI to release the complete PCR product, or with SnaBI as a diagnostic indicator. Digestion with SnaBI should release a fragment of 306 bp (SnaBI 476 to SnaBI 782) that encompasses the CMV promoter. The release of a second fragment that maps to the binding of ITR (SnaBI 142 to SnaBI 476), is contingent upon redispositions that occur during the concatomer formation, and can therefore vary in size of 334 bp (2 full copies of ITR ) up to 0 bp, if the ITRs have been deleted. The PCR product of the 10-3.AV5 cell line thought to contain 1.5 copies of ATR ITR (10-3.AV5), was also cloned into pCRII, and digested with the indicated enzyme. This sample serves as a positive control for digestion by SnaBI for diagnosis. Digestion of this sample with EcoRI correctly releases the 730 bp PCR fragment, as well as a secondary double band of approximately 500 bp in size. It is thought that this secondary band is an artifact due to the secondary structure that occurs in the 1.5 copies of the AAV ITR during replication in bacteria. Digestion of the positive control with SnaBI releases the 306 bp fragment for diagnosis of the CMV promoter, and a 250 bp fragment that maps to the ITR junction. Digestion of the 6 individual clones with EcoRI and SnaBI indicated that deletions of varying lengths occurred in all recovered junctions, and were largely confined to ITRs in the junctions. The analysis of the sequences further indicated that the majority of the deletions spanned portions of both ITRs at the junctions, without involving contiguous viral DNA.

E. Analysis of In Situ Hybridization by Fluorescence (FISH) FISH was performed on skeletal muscle cryoses to characterize the distribution of proviral DNA within the injected tissue. Small muscle pieces of 4 to 5 mm of treated mice or control mice were embedded in OTC and rapidly frozen in liquid isopentane cooled with liquid nitrogen. Frozen sections 10 S thick were cut in a cryomicrotome. The sections were assembled, fixed (Histochoice) and prosed for fluorescence by in situ hybridization using a protocol described above [AND. Gussoni et al., Nat. Biotech. , 14: 1012-1016 (1996)]. Adjacent sections for β-galactosidase activity were collected to identify positive areas for lacZ in bundles of tissue. To quantify the FISH signal, positive areas for lacZ (determined by staining adjacent sections for β-galactosidase activity) were examined under a Nikon FxA microscope for photomicrographs, equipped with epifluorescence. The count of the total number of individual muscle fibers corresponding to the positive area for lacZ was made in the section under a standard mode of phase contrast. The same area was then examined under fluorescence microscopy using the appropriate filter package for rhodamine isothiocyanate. The number of nuclei of the muscle cells showing punctate staining was recorded. Each positive nucleus was examined under phase contrast to investigate if it came from a muscle fiber. For controls, negative areas for lacZ were examined and quantified similarly (areas in the same sections that lacked β-galactosidase activity, or mock sections of transfected muscle). For confocal microscopy, sections were observed under an objective lens oil immersion (100X) on a laser microscope Leica confocal laser-equipped Krypton-Argon (Leica Lasertechnik, GmbH), stations central work for graphic TSC and Voxel View Silicon The images observed under the rhodamine channel were also observed sequentially under differential interference contrast to confirm the location of the fluorescence signal in the muscle cell nuclei. The fluorescence and differential contrast images were then sequentially placed on a central workstation for TCS, and transferred to a Silicon graphics workstation for image processing. The processed images were stored and printed using Photoshop programs. tiAeron sections alternately in series for ß-galactosidase activity to identify muscle fibers expressing the transgene, and hybridized with a biotinylated proviral probe to localize the distribution of the proviral genome. A defined fluorescent seAal was detected in some nuclei of muscle fibers that express β-galactosidase. An analysis of 3 sections in series revealed hybridization in 53/1006 (5.3%) nuclei of fibers that express β-galactosidase, and 0/377 nuclei of fibers that do not express the same. Hybridization was not detected in tissues from uninfected animals (data not shown). The ability to detect viral genomes using FISH, added another dimension to the analysis. Individual hybridization foci were detected in 5% of all nuclei contained within the muscle fibers expressing β-galactosidase. It is possible that this is an erroneous appreciation of the transduced nuclei, due to the sensitivity limitations of this technique, especially for target sequences less than 12 kb in size [B. J. Trask, Trends Genet., 2 = 149-154 (1991)]. The implications of the FISH analysis are interesting. The presence of a proviral genome / diploid myoblast genome, measured by Southern, together with the result of FISH that showed 5% of nucleic acids harboring an AAV genome, would predict that the average concatomer comprises at least ten proviral genomes. These studies show β-galactosidase enzyme activity extending beyond the site of a nucleus transduced by the vector, suggesting an extended nuclear domain of at least 10 Sm. This is consistent with previous investigations that documented extended nuclear domains for cytosolic proteins [H. M. Blau et al., Adv. Exp. Med. & Biol., 280: 167-172 (1990)]. An extended nuclear domain of transgene expression within the syncytial structure is important for gene therapy applications for several reasons. The net yield of recombinant protein from a transduction event may be higher in a syncytium, where the protein distribution is less limited by membrane barriers. In addition, this system has advantages in vector systems that require the expression of multiple recombinant proteins such as those with inducible promoters [J. R. Howe et al., J Biol. Chem., 270: 14168-14174 (1995); V. M. Rivera et al., Nat. Med., 2: 1028-1032 (1996)]. In muscle, the coexpression of recombinant proteins does not require cotransduction with an individual nucleus due to the extensive network of overlapping domains.

EXAMPLE 4 The immune responses directed by the transgene are minimized when using rAAV-free dß helper virus for the release of genes directed towards the muscle.

The stability of lacZ expression in muscle cells achieved from a rAAV vector containing lacZ administered in the absence of helper virus was surprising in light of previous investigations, which demonstrated destructive immune responses generated against β-galactosidase expressed from adenoviral vectors in muscle fibers. We also studied the specific immune responses to the transgene by measuring the levels of anti-β-galactosidase antibodies in serum using Western analysis. Blood was taken from C57BL / 6 and ROSA-26 mice (Jackson Laboratories, Bar Harbor, ME), which were necropsied on day 30 after injection of the virus, and the serum was collected. ROSA-26 is a transgenic line possessing DNA for β-galactosidase from E. coli, and was developed on a 129 basis. Serum was also harvested from C57BL / 6 mice that received an intramuscular injection of recombinant adenoviruses for LacZ (H5. OlOCMVLacZ, 5 x 10 ° pfu in 25 SI of HBS), or the recombinant AAV.CMVLacZ (as described above). Both vectors express E. coli β-galactosidase from a minigene directed by CMV. Aliquots (5 Sg) of purified β-galactosidase from E. coli (Sigma) were resolved on a 10% SDS polyacrylamide gel (5 mg / band), and electrophoretically transferred to a nitrocellulose membrane (Hybond-ECL, Amersham). The blot was incubated with blotto [fat-free milk at 5%, Tris-Hcl at 50 mM (pH 8.0), CaCl at 2 mM and Tween-20 at 0.05%] at room temperature for 2 hours to block the available sites. The individual bands were cut and incubated with serum (diluted 1: 200 in blotto) for 1 hour at room temperature. The location of the antigen-antibody complex was achieved by adding horseradish peroxidase conjugate goat anti-mouse, followed by detection of ECL (Amersham). The cut bands were reassembled on a Mylar sheet before the addition of ECL reagent, and for documentation by film. The intramuscular injection of adenovirus H5. OlOCMVlacZ deleted in El in skeletal muscle of C57BL / 6 mice resulted in the substantial accumulation of antibody to β-galactosidase in serum, which did not occur in identical transgenic animals in MHC that possess an inserted lacZ gene, and which are immunotolerant to β-galactosidase. Significantly, neither the C57BL / 6 mice nor the transgenic animals in lacZ developed antibodies to galactosidase after intramuscular injection of AAV.CMVlacZ.

EXAMPLE 5 Comparative Studies dß Vectors Adßnoviralßs and dß AAV in Muscle Cells Studies on the biology of muscle-directed gene transfer, as measured by adenovirus and recombinant AAV, demonstrate that adenoviruses, but not AAV, infect antigen-presenting cells (APCs), which induce cascading immune responses that lead to destructive cellular and humoral immunity. An experimental paradigm was constructed to define the specific differences in the responses of the host to the gene transfer directed towards the skeletal muscle with adenovirus and recombinant AAV. The aim was to delineate differences in the biology of these vector systems that lead to preferential immunological activation directed against a transgenic product (ie, β-galactosidase) when expressed from a recombinant adenoviral vector, but not an AAV vector. The general procedure was to inject an AAV expressing lacZ in the right leg of a mouse. It has been shown in the previous examples that this confers efficient and stable expression of the gene. In other experimental groups, the animals receive rAAV in addition to various combinations of vectors and cells to define components of the immune response directed against Ad, which lead to destructive cellular and humoral immunity. The effects of these experimental manipulations were followed by the evaluation of their impact on the stability of muscle fibers grafted with rAAV, as well as by the measurement of other immunological parameters. Any intervention that induces ß-gal immunity in muscle fibers can be detected by evaluating the stability of the expression of the transgene in the muscle transduced by AAV, and by the development of inflammation. For this study, four experiméntale groups were developed, as summarized below. The virus was injected into mice using techniques substantially similar to those described in Example 2 above, that is, the virus was suspended in saline regulated at its pH with phosphate, and injected directly into the tibialis anterior muscle. When the animals were necropsied, the muscle tissues were rapidly frozen in isopentane cooled with liquid nitrogen, and cut into 6-m thick sections, while serum samples and draining inguinal lymph nodes were harvested for immunological tests. . Lymphocytes from inguinal lymph nodes were harvested, and a standard chromium (Cr) release test was carried out for 6 hours, essentially as described below, using different effector: target cell ratios (C57SV, H-2b) in 200S1 of DMEM in 96-well plates with a V-shaped bottom. Prior to mixing with the effector cells, the target cells were infected with an adenovirus expressing alkaline phosphatase (AdALP), or stably transduced with a retrovirus expressing lacZ, pLJ -lacZ, labeled with 100 SCi of 51Cr used at 5 x 103 cells / well. After incubation for 6 hours, the count of 100 Si aliquots of the supernatant was counted in a gamma counter. The results for groups 1 to 3 are provided in Figures 5A to 5C. The frozen sections (6Sm) were fixed in methanol, and were stained with anti-CD4 and anti-CD8 antibodies. Morphometric analysis was carried out to quantify the number of CD8 + and CD4 + cells per section. The cytokine release test was carried out essentially in the following manner. The lymphocytes were restimulated for 40 hours with β-galactosidase, purified AAV or adenovirus type 5. The cell-free supernatants (100 SI) were tested for the secretion of IL-10 and IFN-gamma. Proliferation was measured 72 hours later by a pulse of H-thymidine (0.50 SCi / well) for 8 hours. The results for the four groups are provided in Figures 6A and 6B. The test for neutralizing antibody was carried out essentially in the following manner. Mouse serum samples were incubated at 56 ° C for 30 minutes to inactivate the complement, and were then diluted in DMEM in two double steps starting at 1:20. Each dilution of serum (100 Si) was mixed with β-galactosidase or adenovirus type 5. After 60 mini-incubations at 37 ° C, 100 Si of DMEM containing 20% FBS were added to each well. Cells were fixed and ligated for β-galactosidase expression the next day. All cells were stained blue in the absence of serum samples. The results for the four groups are provided in Figures 6A and 6B. The mice of group 1 received AAV.CMVlacZ, produced as described in example 1, on the right leg without any other intervention. Transduction only with AAV.CMVlacZ produced high levels of stable gene transfer (evident even at 28 days) without lymphocyte infiltration. No activation of CD8 T cells was detected (Figure 5). Neither antigen-specific CD4 + T cells were detected [i.e., virus-specific or β-galactosidase (Figures 6A and 6B)]. No antibodies were generated for β-galactosidase or adenovirus (Figs 7A and 7B). Group 2 mice received AAV.CMVlacZ) in the right paw and adenovirus expressing lacZ (H5, OlOCMVlacZ) in the left paw. The objective of this group was to determine if the immune response to the muscle fibers infected with Ad was systemic, as demonstrated by its impact on the biology of the transduced paw by contralateral AAV.CMVlacZ. Apparently, treatment with lacZ adenoviral induced an immune response to β-galactosidase that led to the destruction of the transduced fibers by lacZ from AAV. Not surprisingly, this was associated with the infiltration of CD4 and CD8 T cells into the leg transduced by AAV, and the activation of cytotoxic T lymphocytes for adenoviral antigens and for β-galactosidase (Fig. 8). Activated CD4 T cells specific for AAV, Ad and β-gal antigens and antibodies specific for adenovirus and β-galactosidase were also observed. The animals in group 3 received a mixture of AAV.CMVlacZ and Ad BglII in the right leg. AdBglII is an adenovirus deleted in El that does not express any recombinant gene. The objective of this group was to determine if adenovirus provides an adjuvant effect that would induce immunity to AAV for lacZ in this setting. This did not lead to loss of expression of the transgene, although there was a substantial infiltration of CD8 T cells and some activation of CD4 T cells for the viral antigens, but not for β-galactosidase (Figs 6A-6B). As expected, antibodies were generated towards the adenovirus, but not towards the β-galactosidase (Figs 7A-7B). Animals of group 4 received AAV.CMVlacZ in the right paw, and were adoptively transferred with antigen-presenting cells harvested from unaffected animals and infected ex vivo with the adenovirus. These animals showed a vigorous and effective immune response to β-galactosidase, as demonstrated by loss of transgene expression, and massive infiltration of CD8 and CD4 T cells. The CD4 cells were activated for β-galactosidase in this experiment, as shown in Figures 6A to 6B, and anti-β-galactosidase antibodies were generated, as shown in Figures 7A to 7B.

EXAMPLE 6 Transduction of a purified rAAV vector containing factor IX into skeletal muscle cells, and expression of F.IX at therapeutically useful levels and without inducing a cytotoxic immune response The data provided in this example demonstrate that the method of this invention provides for the prolonged expression of a therapeutic transgene, F.IX, in immunocompetent and immunoincompetent subjects, in the absence of a cytotoxic immune response to transduced cells. In addition, the levels of protein reached in the serum of immunoincompetent animals are adequate to achieve a therapeutic effect. Thus, prolonged expression of F.IX of human in muscle cells by rAAV vectors in immunoincompetent patients, such as those suffering from hemophilia, is useful for releasing F.IX in patients suffering from said disease.

A. Preparation of purified rAAV The rAAV vector used in the following in vivo experiments has an expression cassette containing the human F.IX cDNA that includes a portion of the I intron under transcriptional control of the immediate early gene enhancer / promoter. and the signal of termination of SV40 transcription of cytomegalovirus (CMV). The vector, which contains this expression cassette flanked by AAV ITR sequences, and which completely lacks AAV protein coding sequences, was constructed in the following manner. Recombinant AAV was generated by cotransfection by the cis.IX.IX plasmid (pAAV-FIX) and the pAAV / Ad plasmid acting at the trans position [A. W. Skulimowski and R. J. Samulski, Method. Mol. Genet., 2 = 7-12 (1995)] in 293 human kidney embryonic cells infected with an adenovirus deleted in El, as described by Fisher et al., J. Virol. , 20 = 520-532 (1996). PAAV-FIX was derived from psub201 [Skulimowski and Samulski, cited above], and contains the CMV promoter / enhancer, the human F.IX coding sequence that includes a 1.4 kb fragment of intron I [S. Kurachi et al., J. Biol. Chem., 270: 5276-5281 (1995)], and the SV40 polyadenylation signal, flanked by AAV ITR sequences. The AAV rep and cap gene functions were delivered in the trans position by pAAV / Ad. The deleted adenovirus in El contained a reporter gene for β-galactosidase (LacZ) or for alkaline phosphatase (ALP) to track the potential contamination of rAAV supply materials with this helper virus. The cells were lysed for 48 hours after transfection by sound treatment, and the rAAV particles released were purified by 4 rounds of CsCl density gradient centrifugation, as described by Fisher et al., Cited above. The resulting rAAV-F.IX particles had a density of 1.37-1.40 g / ml. The purified rAAV-F.IX titer was determined by slot blot hybridization using a probe specific for the CMV promoter or plasmid DNA intron I sequences or standards of pAAV-F.IX of known concentration. The ability of rAAV-F.IX to transduce cells in vitro was confirmed by transduction of cultured HeLA cells, and by measuring the concentration of hF.IX in the culture supernatant 36 hours after infection, by an ELISA test specific for hF. IX [J. Walter and others, Proc. Nati Acad. Sci. USA, 93: 3056-3061 (1996)]. RAAV-F.IX (10 ^ 2 ~ i0l3 genomes / ml) was stored at -79 ° C in pH regulated saline with HEPES, pH 7.8, including 5% glycerol. Purified rAAV-F.IX usually lacked detectable amounts of contaminating adenovirus, when analyzed by transduction of 293 cells, followed by staining for alkaline phosphatase or β-galactosidase, as described by Fisher et al., Cited above. Wild type AAV was detected at < 1 infectious unit per 10g genomes of rAAV-F.IX. The wild-type AAV test was as follows: 293 cells cultured on camera slides, coinfected with adenovirus and aliquots of purified rAAV-F.IX, and fixed for immunofluorescence staining 24 hours after infection. A mouse monoclonal antibody against AAV capsid proteins (American Research Products, Belmont, MA) served as a primary antibody, and anti-mouse IgG (DAKO Corporation, Carpinteria, CA) at a dilution of 1:40 as secondary antibody.

B. Introduction dß rAAV in Skeletal Muscle The strains of mice selected for intramuscular injection with rAAV were C57BL / 6 (Charles River Laboratories, Wilmington, MA) and B6, 129 and Rag 1 (Jackson Laboratories, Bar Harbor, Maine). Female mice 4-6 weeks old were anesthetized with an intraperitoneal injection of ketamine (70 mg / kg) and xylazine (10 mg / kg), and a 1 cm longitudinal incision was made in their lower extremity. AAV-F was injected. IX (2X1011 or 1x1010 vector / animal genomes in saline regulated at pH with HEPES, pH 7.8) in the anterior tibialis muscle (25 Si) and the quadriceps muscle (50 Si) of each paw, using a Hamilton syringe. The incisions were closed with Vicryl 4-0 suture. Blood samples were collected at 7-day intervals from the retro-orbital plexus in capillary tubes for microhematocrit, and the plasma was tested for hF.IX by ELISA test (part C below). For immunofluorescence staining (part D below) and DNA analysis (part F below), animals were sacrificed at selected time points, and injected and non-injected muscle tissue was excised. The tissue was placed in compound for OTC inclusion, quickly frozen in isopentane cooled in liquid nitrogen for 7 seconds, and immediately transferred to liquid nitrogen.

Detection of human F.IX dß by dß test ELISA The F.IX antigen of human was determined in mouse plasma by ELISA test, as described by Walter et al., Cited above. This ELISA test showed no cross reaction with mouse F.IX. All samples were evaluated in duplicate. Protein extracts were prepared from the injected muscle of mice, macerating the muscle in pH regulated saline with phosphate (PBS) containing leupeptin (0.5 mg / ml) followed by sound treatment. The cell remnants were removed by microcentrifugation, and 1:10 dilutions of the protein extracts for hF.IX were tested by ELISA. As negative controls extracts of rAV.CMVLacZ (see example 1 above) -injected muscle were used. Protein concentrations were determined by BIORAD test (Bio-Rad, Hercules, CA).

D. Immunofluorescence staining To perform the immunofluorescence staining of tissue sections, muscle tissue (6 Sm) cryosettings were fixed for 15 minutes in 3% paraformaldehyde in PBS, pH 7.4, and rinsed in PBS for 5 minutes. , were incubated in methanol for 10 minutes, washed 3 times in PBS, and then blocked in PBS / bovine serum albumin (BSA) at 3% for 1 hour. Sections were subsequently incubated overnight with an affinity-purified goat anti-human antibody (Affinity Biologicals), so that they were diluted 1: 1000 in PBS / 1% BSA. After 3 washes (10 minutes each) in PBS / 1% BSA, the secondary antibody was applied for 90 minutes (rabbit anti-goat IgG conjugated to FITC, DAKO Corporation, diluted 1: 200 in PBS / BSA to 1%). After three more washes in PBS / 1% BSA, sections were washed in distilled water, air dried and mounted with Fluoromount G mounting media (Fisher Scientific). All incubation steps were carried out at room temperature, except for the incubation with the primary antibody (4 ° C). The same protocol was applied when the sections were stained with rabbit anti-human collagen IV as primary antibody (Chemicon, Temecula, CA) at a dilution at 1: 500 and anti-rabbit IgG conjugated with FITC (DAKO Corporation) as secondary antibody . For colocalization studies, a goat anti-hF.IX antibody conjugated with FITC (Affinity Biologicals) was simultaneously applied with the anti-collagen IV antibody, and rhodamine-conjugated anti-rabbit IgG (Chemicon) was used to detect collagen complexes IV-antibody. Fluorescence microscopy was carried out with a Nikon FXA microscope.

E. Tests for circulating antibody against hF. IX Plasma samples of C57BL / 6 mice injected intramuscularly with AAV-F.IX were tested for the presence of antibodies against hF.IX using the ELISA test. The microtiter plates were coated with human F.IX (ISg / ml in NaHC 3 to 0.1M, pH 9.2). Diluted samples of plasma (1:16) were applied in duplicate, and antibodies against hF were detected. IX with anti-mouse IgG conjugated with horseradish peroxidase (Zymed, San Francisco, CA) at a dilution of 1: 2000. The conditions of the pH regulator were as described in Walter et al., Cited above. The levels of anti-hF. IX were estimated by comparing absorbance values with anti-hF. Mouse monoclonal IX (Boehringer Mannheim) diluted to a final concentration of 1 Sg / ml. Western blots were carried out to demonstrate the presence of anti-hF. IX, as described by Dai et al., Proc. Nati Acad. Sci. USA, 92: 1401-1405 (1995), except that a goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Boehringer Mannheim) was used as the secondary antibody, thus allowing the detection of hF complexes. IX-antibody with ECL reagent (Amersham). The dilution of the mouse plasma was 1: 500.

F. DNA analysis Genomic DNA was isolated from injected muscle tissue, as described for mammalian tissue by Sambrook et al., Molecular Cloning: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). As described in example 3 of the application, PCR reactions were carried out to amplify the double-tailed head-to-tail joints of rAAV. The forward primer 005 [SEQ ID NO: 4] tunes the SV40 polyadenylation signal (position 8014-8033 bp), and the inverted primers 013 [SEQ ID NO: 2] and 017 [SEQ ID NO: 3] come together to the CMV promoter (position 4625-4606 and 4828-4809 bp). PCR reactions were carried out using 100 ng of genomic DNA at a total reaction volume of 10 Si, including MgCl 2 at 1.5 mM and 0.5 SM of primer pair 005/013 or 005/017. After an initial denaturation step (94 ° C for 4 minutes), 35 cycles of the following profile were carried out: denaturation at 94 ° C for 4 minutes, denaturation at 94 ° C for 1 minute, annealing at 52 ° C during 1 minute, and extension at 72 ° C for 90 seconds (10 minutes during the final cycle). The PCR products were cloned (for AD? Sequence analysis) using the T / A cloning kit (Invitrogen, San Diego, CA). Southern blot hybridizations were performed using randomized primed probes labeled with P-dCTP specific for the CMV promoter (for hybridization with PCR fragments) or for intron I of hF.IX, as present in rAAV-F.IX ( for hybridization with mouse genomic DNA).

G. Results of the expression of hF.IX in immunocompetent mice Immunocompetent C57BL / 6 mice were injected intramuscularly with rAAV-hF.IX, and the animals were sacrificed one month after the injection. A test of ELISA on protein extracts of injected muscles (tibialis anterior and quadriceps), demonstrated the presence of 1.8- 2.1 ng of hF.IX / mg of tissue (40-50 ng of hF.IX / mg of protein). The expression of hF.IX in muscle tissues was confirmed by immunofluorescence studies in tissue sections. Immunocompetent C57BL / 6 mice were injected intramuscularly with rAAV-hF.IX, and the animals were sacrificed three months after the injection. Factor IX was not detected in the non-injected muscle. Factor IX was not detected in the muscle injected with a control, rAAV-lacZ. Human expression of F.IX was detected in muscle fibers of C57BL / 6 mice three months after injection (3.3x10 vector genomes per injection site, 200x magnification). Note that F.IX is present not only in the muscle fibers, but also in the interstitial spaces between the fibers, as well as where it seems to accumulate. Interestingly, this staining pattern was identical to that observed with a polyclonal antibody against collagen IV of human, which also t ± Aó the interstitial space. A muscle dye one month after infection with rAAV-hF.IX (3.3 x 10 ° genomes per injection site) with either antibody for collagen IV of human (data not shown). Collagen IV has recently been identified as a binding protein for F.IX of human [W.-F. Cheung et al., Proc. Nati Acad. Sci. USA, 93: 11068-11073 (1996)]. Initial experiments in immunocompetent mice showed that, despite high levels of gene transfer and stable expression of hF.IX in the injected muscle, it was not possible to detect significant quantities of hF.IX in the circulation by ELISA. . See figure 8. Other experiments showed that the animals had developed high titre antibodies against the introduced circulating protein. For example, when the same plasma samples were tested for antibodies to human F.IX, a strong antibody response was observed in all injected animals, starting on day 11 after injection. See Figure 9. Through the use of Western blot analysis, high levels of circulating antibody were found to persist for the duration of the experiment. This finding contrasted with previously reported experience, in which a different administration route, ie, intravenous injection, of a different vector, ie, an adenoviral vector expressing hF.IX, induced a different immune response, i.e. did not trigger the formation of neutralizing antibodies against hF.IX (Walter et al., cited above). However, the levels of protein expression required to induce antibody formation are quite low. The Western blot is a little less sensitive than an ELISA test, but states that there seems to be a rising antibody titer that begins 18 days after the injection. Thus, in immunocompetent animals, the absence of detectable expression of hF.IX at initial time points is a result of the biology of rAAV expression in the muscle, whereas the subsequent absence of detectable F.IX in the circulation results of the production of antibodies for the introduced protein. However, the antibody response in serum was not associated with the cytotoxic immune response directed against the cells expressing the transgene. In fact, no inflammation or extensive tissue damage was observed in any of the tissue sections described above, nor in sections analyzed by H & amp;; E (data not shown). This contrasts with the immune response induced by the injection, in skeletal muscle, of recombinant adenovirus possessing a transgene [Dai et al., Cited above; Y. Yang and others, Hum. Molec. Genet., 5: 1703-1712 (1996); X. Xiao et al., J. Virol. , 70: 8098-8108 (1996)].

H. Expression of df hF.IX in immunodeficient mice AAV-F.IX was released in muscles of Rag 1 mice, which are homozygous for a mutation in gene 1 of recombinase activation. These animals are therefore functionally equivalent to mice with severe combined immunodeficiency (SCID), and do not produce mature B or T cells. A dose of 2x10 rAAV-hF.IX vector genomes per animal resulted in stable expression of hF.IX in mouse plasma. See Figure 10. The F.IX of human was detectable first by ELISA test in the second week after injection, and then increased gradually. Plasma levels in all animals reached a plateau of therapeutic levels of F.IX five to seven weeks after injection at 200 to 350 ng of hF.IX / ml of mouse plasma.

This level was maintained for the duration of the experiment (four months after the injection). When a total of 1x10 ° rAAV-hF.IX vector genomes were injected, expression was three to four times lower, but still reached therapeutic levels (> 100 ng / ml) for some animals. See Figure 11. These levels, which represent 4 to 7% of the normal circulating levels in plasma, are well within a therapeutic range, and demonstrate that the method of this invention is feasible for the treatment of hemophilia, an associated disease with immunodeficiency.

I. DNA analysis results DNA genomic DNA was isolated from muscle tissue injected six to eight weeks after injection. The presence of DNA introduced from the vector was demonstrated by digestion with EcoRV, which releases a 1.8 kb fragment of the vector construct that includes the complete sequence of the 1.4 kb intron I. A probe specific for the hybrid intron I with this fragment, and not cross-hybridized with mouse DNA from an uninjected animal. The undigested DNA appeared as a hybridization signal in the high molecular weight DNA. In addition, PCR primers developed to amplify recombinant AAV tail-to-tail-concator sequences present in transduced cells (Fig. 12) successfully amplified said muscle-isolated DNA sequences from tissue transduced by AAV-F.IX (muscles). tibialis anterior and quadriceps of immunodeficient and immunocompetent animals). The PCR products were visualized by Southern blot hybridization with a probe specific for the CMV promoter / enhancer. The pair of primers 005-013 produced fragments that were 1.0 kb and smaller; primer pair 005-017 amplified fragments that were 1.2 kb and smaller. As expected, these PCR reactions did not result in bands other than the sizes described above, but rather a series of amplification products with a predicted maximum size, due to the imprecise binding of AAV genomes present in these double repeats. [S. K. McLaughlin et al., J. Virol., 62.-1963-1973 (1988)]. The imprecise binding is the result of variable deletions of ITR sequences at the binding sites, as confirmed by the determination of the DNA sequence of cloned PCR products (data not shown).

J. PCR Studies Although arrays of AAV genomes can occur from head to head and tail to tail during viral replication [K. I. Berns, Microbiol. Rev., 54: 316-329 (1990)], head-to-tail arrangements are more typically associated with AAV that has been integrated into the chromosomal DNA of the transduced cell during latent infection [S. K. McLaughlin et al., J. Virol. , 62: 1963-1973 (1988); J. D. Tratschin et al., Mol. Cell Biol., 5: 3251-3260 (1985); N. Muzcyka, Current Topics in Microbioloqy and Immunoloqy, 158: 97-129 (1992)]. Southern blot data on undigested DNA from muscle cells injected with rAAV demonstrated that rAAV DNA derived from host cell genomic DNA six weeks after injection, is present as a kind of high molecular weight. The greater seAal intensity observed with the restricted DNA probably results from an unmasking effect when the fragments are separated from the total genomic DNA [X. Xiao et al., J. Virol. , 20: 8089-8108 (1996)]. This finding, the presence of a hybridization signal for high molecular weight DNA is consistent, as is the case with PCR data, with the integration events that occur during transduction. The integration status of the rAAV described in this paragraph and in paragraph I is also likely to contribute to the stability and prolonged expression of the transgene.

EXAMPLE 7 Expression of ApoE using rAAV administered to a skeletal muscle cell The following example demonstrates the prolonged expression of another transgenic therapeutic product, apolipoprotein E (ApoE), a protein useful in the treatment of atherosclerosis by introducing, in skeletal muscle, a rAAV vector according to this invention. Again, the absence of a destructive CTL response allows the prolonged expression of the transgenic product.

A. Construction of dAl rAAV Recombinant AAV vectors encoding the secreted human protein ApoE were constructed in a manner similar to that described above for F.IX. ApoE cDNA was separated from plasmid pAlterApoE3 (provided by Dr. Rader's laboratory, University of Pennsylvania) by digestion with Xbal, shaved at its ends and cloned into the base structure of pCMVLacZ digested by Notl (see the construction diagram of vector of figure 13). A 2062 bp Smal / Sacl fragment of the lacZ gene in pCMVLacZ was isolated and inserted into the SalI site of the new plasmid as a filler. The minigene cassette for ApoE that now contains the CMV promoter, the SV40 polyadenylation sequences of the ApoE cDNA, and a 2062 bp filler, was isolated by digestion with EcoRI / HindIII (total length: 4.3 kb) and then ligated. to a base structure of pSub201 digested with Xbal. The final product, designated pSubCMVApoE-2062R0, was used as a cis plasmid in the production of rAAV. PoE, following the procedures described above for rAAV. F. IX.

B. In vitro analysis of dE ApoE expression by rAAV.ApoE 84-31 cells seeded in 6-well plates with 2 microliters of rAAV were infected. poE purified with CsCl in 2 ml of Dulbecco's modified Eagle's medium with fetal bovine serum at 2%. The cells were maintained at 37 ° C for 48 hours. An aliquot of the supernatant was then removed from the well for ApoE Western blot analysis. The results showed that the ApoE protein was clearly detectable in the supernatant of 84-31 cells infected with AAV.ApoE.

C. In vivo expression of rAAV.ApoE in mice drugged to express ApoE. 2.5 to 5 x 101 particles of rAAV.ApoE were injected into the tibialis anterior and quadriceps muscles on each side of mice drugged to express ApoE (total particles per mouse. : 5 x 10 10 to 5 x 1011). Prolonged ApoE local expression was detectable by immunofluorescence on days 28 and 120 after injection even in the presence of anti-ApoE antibodies in plasma.

Conclusion This experiment demonstrates that intramuscular injection of the vector, rAAV-ApoE, in mice drugged to express Apo-E leads to muscle fiber transduction and secretion of substantial amounts of the recombinant protein in the circulation. Prolonged expression of the transgene in muscle fiber was achieved in the absence of a destructive immune response of CTL, even though humoral immunity was induced for the secreted protein.

EXAMPLE 8 Expression of trans ßn in a primate A rhesus monkey was anesthetized, its forearm was pinched and aseptically prepared, and a 0.5 cm incision was made in the skin over the anterior tibialis muscle. The fascia was identified, and the viral suspension of rAV.CMVLacZ (described in Example 1) was injected (175 microliters of 10 genomes / ml) at a depth of 5-7 mm in the fascia. Fourteen days later, a muscle biopsy was obtained, frozen in OCT, sectioned and stained in X-gal. The tissue sections were analyzed quantitatively using the Leica Z500MC image processing and analysis system integrated into a Nikon FXA microscope. X-gal histochemistry revealed a high level of β-galactosidase expression in most of the muscle fibers in the area of the injection site. Twenty-four percent of the fibers expressed ß-galactosidase in an area of or 224 mm in the region of the injections. Based on the results described above with ApoE and F.IX, expression is expected to be prolonged in the absence of a cytotoxic immune response. These data support the fact that the expression of a transgene according to this invention can be duplicated in an animal other than a mouse, and particularly in a primate animal.

Numerous modifications and variations of the present invention are included in the specification identified above, and are expected to be obvious to the person skilled in the art. It is believed that such modifications and alterations of the method of the present invention are included in the scope of the appended claims thereto.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Trustees of the University of Pennsylvania. Wilson, James M. Fisher, Krishna J. (Ü) TITLE OF THE INVENTION: Method for gene therapy directed by recombinant adeno-associated virus (iii) NUMBER OF SEQUENCES: 4 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Howson and Howson (B) STREET: Spring House Corporate Cntr, PO Box 457 (C) CITY: Spring House (D) STATE: Pennsylvania (E) COUNTRY: UNITED STATES OF NORTH AMERICA (F) POSTAL CODE: 19477 (v) LEGIBLE COMPUTER FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER : compatible with IBM PC (C) OPERATING SYSTEM: PC-DOC / MS-DOS (D) PROGRAMS: Patentln Relay - # 1.0, Version # 1.30 (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: WO (B) ) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) PREVIOUS DATA OF THE APPLICATION: (A) APPLICATION NUMBER: US 08 / 708,188 (B) DATE OF SUBMISSION: 06-SEPT-1996 (vii) PREVIOUS DATA OF THE APPLICATION : (A) APPLICATION NUMBER: US 08 / 729,061 (B) DATE OF SUBMISSION: 10-OCT-1996 (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Kodroff, Cathy A. (B) REGISTRATION NUMBER: 33,980 (C) REFERENCE / CASE NUMBER: GNVPN.019CIP2PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 215-540-9200 (B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ ID NO.-l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10398 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: l (Xi) EESCRIHIECKEE SEQUENCES: SEQ ID NO: l: GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT ATGATAATGA 60 GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG GGCGGGTGAC GTAGTAGTGT 120 GGCGGAAGTG TGATGTTGCA AGTGTGGCGG AACACATGTA AGCGACGGAT GTGGCAAAAG 180 TGACGTTTTT GGTGTGCGCC GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC 240 GGATGTTGTA GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT 300 GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA ATATTTGTCT 360 AGGGAGATCT GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC 420 GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA 480 ACTCCATCAC TAGGGGTTCC TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA 540 TTCGAGCTTG CATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600 CCGCCCAACG ACCCCCCCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 660 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA 720 GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 780 CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTC CTACTTG GCAGTACATC 840 TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900 GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 960 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 1020 ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 1080 AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG 1140 GACCGATCCA GCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAACTGAA AAACCAGAAA 1200 GTTAACTGGT AAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC GGTGGTGGTG 1260 CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT TCTAGGCCTG TACGGAAGTG 1320 TTACTTCTGC TCTAAAAGCT GCGGAATTGT ACCCGCGGCC GCAATTCCCG GGGATCGAAA 1380 GAGCCTGCTA AAGCAAAAAA GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA 1440 TTTTCGTTGC CGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500 ATCCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC 1560 TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC 1620 CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCTT TGCCTGGTTT C CGGCACCAG 1680 AAGCGGTGCC GGAAAGCTGG CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC 1740 CCTCAAACTG GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800 TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC TCGCTCACAT 1860 TTAATGTTGA TGAAAGCTGG CTACAGGAAG GCCAGACGCG AATTATTTTT GATGGCGTTA 1920 ACTCGGCGTT TCATCTGTGG TGCAACGGGC GCTGGGTCGG TTACGGCCAG GACAGTCGTT 1980 TGCCGTCTGA ATTTGACCTG AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA 2040 TGGTGCTGCG TTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100 GCATTTTCCG TGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC GATTTCCATG 2160 TTGCCACTCG CTTTAATGAT GATTTCAGCC GCGCTGTACT GGAGGCTGAA GTTCAGATGT 2220 GCGGCGAGTT GCGTGACTAC CTACGGGTAA CAGTTTCTTT ATGGCAGGGT GAAACGCAGG 2280 TCGCCAGCGG CACCGCGCCT TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTATGCCG 2340 ATCGCGTCAC ACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400 ATCTCTATCG TGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT GAAGCAGAAG 2460 CCTGCGATGT CGGTTTCCGC GAGGTGCGGA TTGAAAATGG TCTGCTGCTG CTGAACGGCA 2520 AGCCGTTGCT GATTCGAGGC GTTAACCGTC ACGAGCATCA TCCTCTGCAT GGTCAGGTCA 2580 TGGATGAGCA GACGATGGTG CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG 2640 TGCGCTGTTC GCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700 TGTATGTGGT GGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG AATCGTCTGA 2760 CCGATGATCC GCGCTGGCTA CCGGCGATGA GCGAACGCGT AACGCGAATG GTGCAGCGCG 2820 ATCGTAATCA CCCGAGTGTG ATCATCTGGT CGCTGGGGAA TGAATCAGGC CACGGCGCTA 2880 ATCACGACGC GCTGTATCGC TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG 2940 AAGGCGGCGG AGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000 ATGAAGACCA GCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG CTTTCGCTAC 3060 CTGGAGAGAC GCGCCCGCTG ATCCTTTGCG AATACGCCCA CGCGATGGGT AACAGTCTTG 3120 GCGGTTTCGC TAAATACTGG CAGGCGTTTC GTCAGTATCC CCGTTTACAG GGCGGCTTCG 3180 TCTGGGACTG GGTGGATCAG TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG 3240 CTTACGGCGG TGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300 TCTTTGCCGA CCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG CAGCAGTTTT 3360 TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG CGAATACCTG TTCCGTCATA 3420 GCGATAACGA GCTCCTGCAC TGGATGGTGG CGCTGGATGG TAAGCCGCTG GCAAGCGGTG 3480 AAGTGCCTCT GGATGTCGCT CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC 3540 AGCCGGAGAG CGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600 CATGGTCAGA AGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG GAAAACCTCA 3660 GTGTGACGCT CCCCGCCGCG TCCCACGCCA TCCCGCATCT GACCACCAGC GAAATGGATT 3720 TTTGCATCGA GCTGGGTAAT AAGCGTTGGC AATTTAACCG CCAGTCAGGC TTTCTTTCAC 3780 AGATGTGGAT TGGCGATAAA AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG 3840 CACCGCTGGA TAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900 TCGAACGCTG GAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG CAGTGCACGG 3960 CAGATACACT TGCTGATGCG GTGCTGATTA CGACCGCTCA CGCGTGGCAG CATCAGGGGA 4020 AAACCTTATT TATCAGCCGG AAAACCTACC GGATTGATGG TAGTGGTCAA ATGGCGATTA 4080 CCGTTGATGT TGAAGTGGCG AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC 4140 AGCTGGCGCA GGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200 CCGACCGCCT TACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA GACATGTATA 4260 CCCCGTACGT CTTCCCGAGC GAAAACGGTC TGCGCTGCGG GACGCGCGAA TTGAATTATG 4320 GCCCACACCA GTGGCGCGGC GACTTCCAGT TCAACATCAG CCGCTACAGT CAACAGCAAC 4380 TGATGGAAAC CAGCCATCGC CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG 4440 ACGGTTTCCA TATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500 TACAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA TAATAATAAC 4560 CGGGCAGGCC ATGTCTGCCC GTATTTCGCG TAAGGAAATC CATTATGTAC TATTTAAAAA 4620 ACACAAACTT TTGGATGTTC GGTTTATTCT TTTTCTTTTA CTTTTTTATC ATGGGAGCCT 4680 ' ACTTCCCGTT TTTCCCGATT TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG 4740 GTATTATTTT TGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800 TTTCTGACAA ACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT GATAAGATAC 4860 ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA 4920 ATTTGTGATG CTATTGCTTT ATTTGTAACC. ATTATAAGCT GCAATAAACA AGTTAACAAC 4980 AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT 5040 CCTCTAGAGT CGAGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT 5100 AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 5160 AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG 5220 CAGATCTGGA AGGTGCTGAG GTACGATGAG ACCCGCACCA GGTGCAGACC CTGCGAGTGT 5280 GGCGGTAAAC ATATTAGGAA CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC 5340 GATCACTTGG TGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400 TGAGGTACTG AAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA AGGTGGGGGT 5460 CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG CCATGAGCAC CAACTCGTTT 5520 GATGGAAGCA TTGTGAGCTC ATATTTGACA ACGCGCATGC CCCCATGGGC CGGGGTGCGT 5580 CAGAATGTGA TGGGCTCCAG CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC 5640 TTGACCTACG AGACCGTGTC TGGAACGCCG TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700 GCCGCTGCAG CCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG CCCGCTTGCA 5760 AGCAGTGCAG CTTCCCGTTC ATCCGCCCGC GATGACAAGT TGACGGCTCT TTTGGCACAA 5820 TTGGATTCTT TGACCCGGGA ACTTAATGTC GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG 5880 CAGGTTTCTG CCCTGAAGGC TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA 5940 CCAGACTCTG TTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000 CGCGCGCGGT AGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG TATTTTTTCC 6060 AGGACGTGGT AAAGGTGACT CTGGATGTTC AGATACATGG GCATAAGCCC GTCTCTGGGG 6120 TGGAGGTAGC ACCACTGCAG AGCTTCATGC TGCGGGGTGG TGTTGTAGAT GATCCAGTCG 6180 TAGCAGGAGC GCTGGGCGTG GTGCCTAAAA ATGTCTTTCA GTAGCAAGCT GATTGCCAGG 6240 GGCAGGCCCT TGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTCGG 6300 GATATGAGAT GCATCTTGGA CTGTATTTTT AGGTTGGCTA TGTTCCCAGC CATATCCCTC 6360 CGGGGATTCA TGTTGTGCAG AACCACCAGC ACAGTGTATC CGGTGCACTT GGG AATTTG 6420 TCATGTAGCT TAGAAGGAAA TGCGTGGAAG AACTTGGAGA CGCCCTTGTG ACCTCCAAGA 6480 TTTTC CATGC ATTCGTCCAT AATGATGGCA ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG 6540 ATATTTCTGG GATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600 TTTACAAAGC GCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC CGGCCCAGGG 6660 GCGTAGTTAC CCTCACAGAT TTGCATTTCC CACGCTTTGA GTTCAGATGG GGGGATCATG 5720 TCTACCTGCG GGGCGATGAA GAAAACGGTT TCCGGGGTAG GGGAGATCAG CTGGGAAGAA 6780 AGCAGGTTCC TGAGCAGCTG CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT 6840 ACCGGGTGCA ACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900 ACTTCGTTAA GCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC CAG AGGCGC 6960 TCGCCGCCCA GCGATAGCAG TTCTTGCAAG GAAGCAAAGT TTTTCAACGG TTTGAGACCG 7020 TCCGCCGTAG GCATGCTTTT GAGCGTTTGA CCAAGCAGTT CCAGGCGGTC CCACAGCTCG 7080 GTCACCTGCT CTACGGCATC TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC 7140 TTTCGCTGTA CGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200 GGCGCAGGGT CCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT CCGGGCTGCG 7260 CGCTGGCCAG GGTGCGCTTG AGGCTGGTCC TGCTGGTGCT GAAGCGCTGC CGGTCTTCGC 7320 CCTGCGCGTC GGCCAGGTAG CATTTGACCA TGGTGTCATA GTCCAGCCCC TCCGCGGCGT 7380 GGCCCTTGGC GCGCAGCTTG CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT 7440 TGAGGGCGTA GAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500 AGGCCCCGCA GACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT TCGGGGTCAA 7560 AAACCAGGTT TCCCCCATGC TTTTTGATGC GTTTCTTACC TCTGGTTTCC ATGAGCCGGT 7620 GTCCACGCTC GGTGACGAAA AGGCTGTCCG TGTCCCCGTA TACAGACTTG AGAGGCCTGT 7680 CCTCGACCGA TGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC 7740 ATGACTATCG TCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800 CCGGCAGCGC TCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC GACGATGATC 7860 GGCCTGTCGC TTGCGGTATT CGGAATCTTG CACGCCCTCG CTCAAGCCTT CGTCACTGGT 7920 CCCGCCACCA AACGTTTCGG CGAGAAGCAG GCCATTATCG CCGGCATGGC GGCCGACGCG 7980 CTGGGCTACG TCTTGCTGGC GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT 8040 CTTCTCGCTT CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100 GATGACGACC ATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG CCTAACTTCG 8160 ATCACTGGAC CGCTGATCGT CACGGCGATT TATGCCGCCT CGGCGAGCAC ATGGAACGGG 8220 TTGGCATGGA TTGTAGGCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT 8280 GCATGGAGCC GGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA 8340 CCACTCCAAG AATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400 CTTGGCAGAA CATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG CGCATCTCGG 8460 GCAGCGTTGG GTCCTGGCCA CGGGTGCGCA TGATCGTGCT CCTGTCGTTG AGGACCCGGC 8520 TAGGCTGGCG GGGTTGCCTT ACTGGTTAGC AGAATGAATC ACCGATACGC GAGCGAACGT 8580 GAAGCGACTG CTGCTGCAAA ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT 8640 TCCGTGTTTC GTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700 TCTGCATCGC AGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA TTAACGAAGC 8760 CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 8820 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 8880 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 8940 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 9000 GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 9060 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 9120 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 9180 TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 9240 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 9360 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 9420 ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 9480 CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 9540 AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG 9660 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 9720 GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 9780 GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTC? TGGTTA TGGCAGCACT GCATAATTCT 9840 CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT 9960 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 10020 AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 10080 AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 10140 CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200 CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 10260 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 10320 1 CCTGACGTCT AAGAAACCAT ATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG 10380 AGGCCCTTTC GTCTTCAA 10398 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CATGGTAATA GCGATGACTA (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3 GCTCTGCTTA TATAGACCTC (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 ATAAGCTGCA. ATAAACAAGT

Claims (9)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a recombinant adeno-associated virus (rAAV) comprising a heterologous gene operably linked to sequences that control the expression thereof in a cell for the manufacture of a medicament for reducing the immune response to rAAV, wherein the rAAV is substantially free of contamination with a helper virus and is administers to a skeletal muscle cell.

2. The use of the recombinant adeno-associated virus (rAAV) comprising a transgene operably linked to sequences that control the expression thereof in a cell for the manufacture of a medicament for prolonging the expression of the transgene, wherein the rAAV is substantially free of contamination with a helper virus and is administered to a skeletal muscle cell.

3. The use according to claim 1 or 2, wherein the transgene is a secretable protein.

4. The use according to claim 3, wherein the protein is selected from the group consisting of Factor IX, ApoE, β-interferon, insulin, erythropoietin, growth hormone and parathyroid hormone.

5. The use according to any of claims 1 to 4, wherein the rAAV consists, from the 5 'end to the 3' end, of inverted terminal repeats (ITRs) of 5 'AAV, a heterologous promoter, the transgene, a polyadenylation sequence and 3 'AAV ITRs.

6. The use according to claim 1 or 2, wherein the transgene is a dystrophin gene.

7. - A method for expressing a transgene in a skeletal muscle cell in the absence of a cytotoxic immune response directed against the cell, characterized in that it comprises the step of introducing into the cell a recombinant adeno-associated virus (rAAV) comprising an operably linked transgene to sequences that control their expression, wherein the rAAV is substantially free of contamination with a helper virus, and wherein the transgene is expressed in the cell.

8. - The method according to claim 7, further characterized in that the transgene is a secretable protein.

9. - The method according to claim 8, further characterized in that the protein is selected from the group consisting of Factor IX, ApoE, β-interferon, insulin, erythropoietin, growth hormone and parathyroid hormone. 10.- The method according to the claim 7, further characterized in that the rAAV consists, from the 5 'end to the 3' end, of inverted terminal repeats (ITRs) of AAV 5 ', a heterologous promoter, the transgene, a polyadenylation sequence and 3' AAV ITRs.