MXPA97003105A - Hybrid adenovirus-virus aav and mi method of use - Google Patents

Abstract

The present invention provides a hybrid vector construct consisting of a portion of an adenovirus, and 5 'and 3' ITR sequences of an AAV and a selected transgene. An interlaced hybrid virus is also provided through a polycation conjugate to a rep AAV gene to form a single particle. These transinfection particles are characterized by a high concentration of transgene assortment to a host cell and by the ability to stably integrate the transgene into the chromosome of the host cell, the use of hybrid vectors and viruses to produce large quantities is also described. of AAV recombine

Description

HYBRID OF ENVIRUS-VIRUS FLUV AND METHOD OF USE This invention was supported by the National Institute of Alu to No. Of concession P30 DK 47757. FJJ1 US government has rights over is-invention » CñMPQ DE Lñ INVENCIÓN The present invention relates to the "arn of the vectors useful in somatic gene therapy and their production.

BACKGROUND OF THE INVENTION The adenoviruses are able to provide extremely high levels of transgene delivery v ertically to all cell types, depending on their myotic status. It can easily generate high titers C 1013 plaque-forming units / ml) of recombinant virus in 293 cells (the equivalent of adenovirus to the retrovirus packaging cell lines) and can be stored for a prolonged period, without loss appreciable. The primary limitation of this virus as a resi.de vector in the complexity of the adenovirus genome. A human adenovirus consists of a linear, double-stranded ODN genome of about 36 lb, which is divided into 100 map units (u.rn.), each of which has a map units (u. rn.), each of which has a length of 360 base pairs (bp). The flDN contains short inverted terminal (RTT) repeats at each end of the genome, which are necessary for the reproduction of viral PON. Gene products are organized in early (El to E4) and late (Ll to L5) regions, based on the expression before or after the start of viral ODN synthesis [See, for example, Horwitz, V rology , 2a. edition, editor B. N. Eields, Raven Press, Itd-, New York (1990) 1. A human adenovirus is subjected to a highly regulated program during its normal viral life cycle TY. Yang and co-authors, Proc. Nati, flcad. ci. USft, 91: 4407-4 1 (1994) 3. Vi nons are internalized by endocytosis mediated by the receptor and transported to the nucleus, where the immediate early genes Fia and Elb are expressed. Because these early gene products regulate the expression of a variety of host genes (which sensitize the cell to the production of viruses) and are central to the cascade activation of early-retarded genes (eg, E2, E3 and E4), followed by late genes (for example, Ll-5), the first generation of recombinant adenoviruses for gene therapy focused on the elimination of the El domain. This strategy was successful in rendering the reproduction of vectors defective; however, the mv vo studies revealed that the expression transgenetioa was transient and was invariably associated with the development of severe inflammation at the target site of the CS vector. Ishibashi and co-authors, J. Clin. Invest., 93: 1885-1893 (1994); 3. M. Uil are and coauthors, Proc. Nati Picad. Sci-, U fí, 35: 4421-4424 (1988); Jl. M. Uilson and coauthors, Clin. Bio., 3: 21-26 (1991); M. Grossman and co-authors, Soin C l 1 and Mol.

Gen., 17: 601-607 (1991) 3. Adeno-associated viruses (VñP) have also been used as vectors. Vñfi is a small, single-sowed (nd) ODN virus with a simple geometric organization (4.7 kg) that makes it an ideal substrate for genetic engineering. Two open reading frames encode a series of polypeptides re and cap. Rep. Polypeptides (re? 78, rep68, re? 62 and rep40) are involved in the reproduction, rescue and integration of V? I? S genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the open reading frames rep and cp, at the 5 'and 3' ends, are the inverted terminal repeats (RTI) of 145 bp, whose first 125 bp are capable of forming duplex structures in the form of Y or T. Ee It is important for the development of Vññ vectors that all rep and c domains can be excised and replaced by a therapeutic transgene or CB reporter. 3. Cárter in Handbook of Parvoviruse, ed. P.

Ti sser, CRC Press, pages 155-168, (1990)]. It has been shown that RTIs represent the minimum sequence necessary for the reproduction, rescue, packaging and integration of the Vññ genome.

The life cycle of the Vññ is biphasic, composed of latent episodes and lit co. During a latent infection, Vñfi vipons enter a cell, co or an encapsidated ssfiDN and shortly thereafter they are intruded into the nucleus, where the Vññ RON is stably integrated into a host chromosome, with no apparent need for division. of the host cell. In the absence of the auxiliary virus1, Vññ's genoin integrated into the ssüüN remains dormant, but is capable of being activated and rescued. The litica phase of the life cycle starts < . uan or a cell harboring a Vfiñ provirus is challenged with a secondary infection by a herpes virus or an adenovirus encoding helper functions, which are recruited by Vñfi to assist in its division of the chromium host (B. Crankcase - cited backwards). The parental infection ssfiDN expands into the reproductive (FR) duplex form of ñDN in a form that depends on rep. The rescued Vfifl genomes are packaged into preformed protein capsules (icosahedral symmetry, approximately 20 nm in diameter) and released as infectious viruses that have more or less ssfiDN genomes packed, after cell lysis. Progress towards the establishment of the Vfi fi as a transducer vector for gene therapy has been slow for a variety of reasons. Although Vfifi's ability to integrate into quiet cells is important, in terms of the long-term expression of a potential transducer gene, the tendency of the integrated provirus to become preferentially involved - "only to specific sites on chromosome 19 reduces its utility. However, there are difficulties surrounding the large scale production of recornbinants with reproductive defect In contrast to the production of recombinant retroviruses or adenoviruses, the only widely recognized means to manufacture vinifers from Vfifl transducers involves cotransfection with two different plasmids Although one of them contains the therapeutic rninigene or informant walled between two VTIn RTIs that act on the Vfifi components that are necessary for the rescue and the subsequent packaging of the recombinant genomes is of progeny, they are provided in ran by a second plasmid that encodes open reading frames Viral proteins for proteins rep and cap. The target cells for the transfection also must be infected with adenovirus, thus providing the necessary auxiliary functions. Because the performance of the Vfifi re com bina rite depends on the number of cells that are transfected with the plasmids of action in cis and in ans, it is convenient to use a transfectation protocol of high efficiency. However, for large-scale production of high-grade viruses, the highly efficient systems of calcium phosphate and liposome, previously used, are laborious and subject to inconsistencies. There remains a need in the art to develop 25 vectors that solve the disadvantages of known vector systems.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides an adenovirus / Vññ recombinant hybrid virus comprising an adenovirus capsid containing selected portions of an adenovirus sequence, Vññ 5 'and 3' RTI sequences, which flank a selected transgene, under the control of a selected promoter and other conventional vector regulatory components. This hybrid virus is characterized by the delivery of high titer transgenes to a host cell and by the ability to stably integrate the transgene into the chromosome of the host cell, in the presence of the rep gene. In one modality, the transgene is a reporter gene. Another modality of the hybrid virus contains a therapeutic transgene. In a preferred embodiment, the hybrid virus has an ion sequence associated with it and the rep gene of Vfifl. This construction is called the hybrid virus conjugate or the trans-infection particle. In another aspect, the present invention provides a hybrid vector construct for use in the production of the hybrid virus or a viral particle described above. This hybrid vector comprises selected portions of an adenovirus sequence, the 5 'and 3' sequences of VTfl RTT, which flank a selected transgene, under the control of a selected promoter and other conventional vector regulatory components.

In another aspect, the invention provides a composition that compresses a hybrid viral particle for use in the delivery of a selected gene to a host cell. Said composition can be used to surn? N a therapeutic gene to a target host cell, to treat or correct an alteration or genetically associated disease. In yet another aspect, the present invention provides a method for producing the hybrid virus by transfecting a suitable packaging cell line, < on the construction of the hybrid vector of this invention. In another embodiment, the method comprises comparing a cell line (either a packaging cell line or a non-packaging cell line) with a hybrid vector construct and a suitable helper virus. In yet another aspect, the present invention provides a method for producing large quantities of Vfifi recornbinant particles, with great efficiency, employing the above methods, which employ the hybrid vector construction of this invention, and collecting rVfifi particles from a line of packaging cells transfected with the vector *. Other aspects and advantages of the present invention are further described in the following detailed description of its preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS The Iñ diagram is a schematic diagram of a vector construction pfid.ñV.CMVLacZ (SEO TD NO: 1), which contains (in dextrorotatory order from above) the adenovirus sequence map, units or ~ l (clear bar ); the 5 'RTT of Vfifi (solid bar); an early inereminator / romotor immediate to CMV (hachurada arrow), an SV40 intron (clear bar), a cflDN (LacZ) of beta-galactosi dasa from E. coll, a SV40 polyamide signal (clear bar, a RTI 3 ' of Vññ (solid bar) the adenovirus sequence of the map units 9-16 (light bar) and a portion of the plasmid derived from? BR322 (thin solid line) .The restriction endonuclease enzymes are identified by their conventional designations, and the location of each restriction enzyme is the identification by * the number of nucleotides in parentheses, to the right of the designation of the enzyme, Figure IB is a schematic drawing demonstrating the linearization of pfid. ñV.CMVLacZ (SEQ ID NO: 1) by digestion with a restriction enzyme NHel and a linear arrangement of an adenovirus type 5, digested with Clal, with omissions of u.rn. 0-1. homologous recombination (between u.rn. 9-16). The plasmids, as in those of adenovirus, are indicated by crossed lines. Figure IC is a schematic drawing showing the hybrid virus ñd.ñV.CMVLacZ, after cotransfection of the linearized pfid.nv.CMVLacZ (SEQ ID NO: 1) and the adenovirus in 293 cells, followed by recornbi naci n homologa mt racel? Lar. FIGS. 2ñ- ?K inform the ODN upper filament of the double filament plasmid pRd.fiV.CMVLacZ (SEQ ID NO: 1) (the complementary filament can be easily derived by one skilled in the art). With reference to SEQ ID NO: 1, nucleotides 1-365 are 5 sequences of the adenovirus type; the Vfifi RTI sequence in 5 'covers the nucleotides 366-538; the micromotor / i creator of CMV includes nucleotides 563-1157; the? ntr * on SV-40 covers nucleotides 1158-1179; the LacZ gene extends to nucleotides 1356-4827; the polyfine sequence SV-40 includes the nucleotides 4839-5037; the RTI of 3 'VRfi includes nucleotides 5053 a 5221; Nucleosides 5221 to about 8100 are the type 5 sequences of adenovirus. The remaining sequences are non-specific plasmid sequences. Figure 3 is a bar graph plotting the ultraviolet absorbance at 420 nm of the blue color of beta-galac-tosidase for one control, and ten positive putative clones (Dlñ to DIO) of 293 cells transfected with the recombinant hybrid ñdflV.CMVLacZ . Eight of the clones expressed high levels of enzyme. Figure 4 is a schematic diagram of? Rep78 / 52 (SEQ ID NO: 2). This plasmid includes a P5 promoter from Vflfi, Rep78, Re? 52 and a poly-fl sequence in a plasmid background PUC18. in Figures 5fi ~ 5E report nucleotides 1-4910 of the upper flnd filament of the double-stranded plasmid pRe? 78/52 (SEQ ID NO: 2) (the complementary filament can be easily derived by one skilled in the art. matter). Figure 6 is a flow diagram of the construction of a transfection particle, formed by a hybrid virus, a poly-L-lysine sequence and the coupled plasmid containing rep from Vfifi. Figure 1 is a flow diagram of the hybrid virus life cycle, in which a particle of transfection enters the cell and is transported to the nucleus. The virus is discovered and the rescue day of the inserted gene, which is then integrated into the coma of the host cell.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a single gene transfer vehicle, which solves many of the limitations of viral vectors of the prior art. This hybrid virus, formed by genetic engineering, contains selected adenovirus domains and selected Vfifi domains, as well as a selected transgene and regulatory elements in a viral capsid. This novel hybrid virus solves the problems observed with other conventional viruses for gene therapy, because it is characterized by the ability to provide extremely high levels of transgene delivery, particularly to all types of cells (conferred by their sequence). adenovirus) and the ability to provide long-term, stable transgenee integration in the host cell (conferred by its Vññ sequences). The hybrid adenovir-s virus of this invention has utility both as a novel vehicle for gene transfer, and as a reagent in a method for the production of recombinant virus, on a large scale. In a preferred embodiment, a transfection or hybrid virus conjugate particle, composed of the fid / Vfifi hybrid virus, conjugated to a rep expression plasmid by means of a poly-lysine bridge is provided. This transfection particle is advantageous because the adenovirus carrier * can grow to sufficient titers for high MOI infections of a large number of cells; the adenoviral genome is efficiently transported to the nucleus in non-dividing cells, such as a complex that facilitates transduction to mitotic cells at rest, and the incorporation of the rep plasmid in the transfection particle provides a high but transient expression of rep , which is necessary both for the rescue of the RVFIFL fiDN and for the efficient and specific integration for the customer.

I. CONSTRUCTION OF THE HYBRID VECTOR AND THE VIRUS fi THE COMPONENT FLDENOVIRUS OF VECTOR AND VIRUS The hybrid virus of the invention uses adenovirus nucleic acid sequences as a shuttle to deliver a Vfifi recornbinant / transgene genome to a target cell. The ñDN sequences of many types of adenovnus, including ti or ñdf. , etan available from Genbank- (gene bank). The adenovirus sequences can be * obtained from any known type of adenovirus, including the 41 currently typed human types (Horwi z and coauthors, previously cited). Similarly, adenoviruses known to infect other animals may be employed in the vector constructions of this invention. The selection of the type of adenovirus is not anticipated as a limitation of the following invention. A variety of adenovirus strains are available from the Firnecan Type Culture Collect ion, Rockville, Maryland, E.U.fl., or can be obtained upon request from a variety of commercial and institutional sources. In the following exemplary embodiment, an adenovirus type 5 (fld5) is used for convenience. The adenovirus nucleic acid sequences employed in the hybrid vector of this invention can vary from a minimum sequence amount, which requires the use of an helper virus to produce the hybrid virus particle, c * only selected omissions of adenovirus genes; said omitted gene products can be supplied in the hybrid viral production process by a selected packaging cell. Specifically, at least the adenovirus nucleic acid sequences employed in the pfidD transfer vector of this invention are adenovirus gene sequences from which all viral genes have been omitted and which contain only those adenovirus sequences necessary for pack the Adenov ral genomic DNA in a preformed capsid head. More specifically, the adenovirus sequences used are the inverted terminal repeat (RTI) sequences 5 'and 3' which act in cis, of an adenovirus (which function as origins of reproduction) and the epator / enhancer domain. 5 'native, which contains sequences necessary to pack the linear fld genomes and the elements increases or is for the El promoter. According to this invention, the entire 5' sequence of adenovirus containing the 5 'RTT and the packaging / incrementing region it can be used as the 5 'sequence of adenovirus in the hybrid virus. This left terminal sequence (5 ') of the fld5 genome used in this invention extends to base pairs 1 to 360, approximately, of the conventional adenovirus genome, also known as apa 0-1 units of the genome. viral, and generally it has a length of 353 to 360 nucleotides, approximately. This sequence includes the 5 'RTI (pb 1-103 of the adenovirus genome) and the comparator / multiplier domain (bp 194-358 of the adenovirus genome). Preferably this 5 'region of the native adenovirue is used in the hybrid virus and the vec tor in unmodified form, filteraily, can be replaced by * corresponding sequences from other types of adenovirus. These fid sequences can be modified to contain the desired omissions, substitutions or mutations, provided that < All the desired function is not eliminated. The 3 'adenovirus sequences of the hybrid virus include the RTI sequence of the right terminal (3') of the adenoviral genome, which extends to base pairs 35,353 until the end of the adenovirus genome, or map units around of 98.4-100. This sequence has a length generally of about 580 nucleotides. All this sequence is conveniently employed as the 3 'sequence of a hybrid virus. Preferably, the 3 'region of native adenoviruses in the hybrid virus is used in unmodified form. However, as described above with respect to the 5 'sequences, some modifications to those sequences, which do not adversely affect their biological function, may be acceptable. As described below, when such 5 'and 3' sequences of adenovirus are employed in the hybrid vector, an auxiliary adenovirus is needed that delivers all the extra genes essential for viral formation, either alone or with a packaging cell line , in the production of the hybrid virus or the viral particle. Alternative embodiments of the hybrid virus employ adenovirus sequences in addition to the minimal sequences, but contain omissions of the -totality or portions of the adenovirus genes. For example, the immediate adenovirus to the early gene Ela (which extends from u.rn 1.3 to u.5) and the early gene omitted Elb (which extends from the 4.6 to the um 11.2), they should be omitted from the adenovirus sequence that is part of the hybrid vector and virus const uct. Alternatively, if those sequences are not completely eliminated, at least a sufficient portion of the Ela and Elb sequences must be omitted, in order to render virus reproduction defective. These omissions, whether complete or partial, that eliminate the biological function of the gene, are referred to here as "functional omissions". Physically, all or a portion of the early, delayed adenovirus E3 gene (which extends from U.S. 76.6 to U.RN 86.2) can be deleted from the adenovirus sequence that is part of the hybrid virus. The function of E3 is irrelevant to the function and production of the hybrid virus. The whole or a portion of the temperate gene E2a, delayed, of the adenovirus (extending from u.m. 67.9 to 61.5) can be eliminated from the hybrid virus. It is also anticipated that portions of the other early delayed genes E2b (ranging from um? 9 to u.rn. 14.2) and E4 (ranging from u.rn.96.8 to um 93.3) may also be eliminated from the hybrid virus and the vector. Omissions may also occur in any of the late genes Ll to L5, which extend from the u.m. 16.45 to 99 of the adenovirus genome. Similarly, omissions may be useful in intermediary genes IX that map between the u.m. 9.8 and 11.2 of! Va2, which form the map between 16.1 and 11.1. Others or sione-- may have occurred in other structural adenoviruses or are not structural. The omissions discussed above may occur individually, i.e., an adenovirus sequence for use in the present invention may contain omissions only. Alternatively, omissions of all genes or effective portions may occur to destroy or biological activity, in any combination. For example, in an exemplary hybrid vector, the adenovirus sequence may contain omissions of the El and E3 genes, or of the El, E2a and E3 genes, or of the El and E4 genes, or of the Ei, E2a genes and E4, with or without omission of E3, etc. The more omissions are made in the adenovirus sequence up to the minimum sequences identified above, which characterize the hybrid virus, the greater the sequences of the components described later, which are to be inserted into the hybrid vector. As described above for the minimal adenovirus sequences, those gene sequences not present in the adenovirus portion of the hybrid virus must be supplied either by a packaging cell line and / or by an auxiliary adenovirus, to generate the I saw hybrid rus. In an exemplary hybrid virus of this invention, which is described below and in Example 1, the genomic sequences of adenovirus present are from urn 0 to 1, um 9 to 78.3 and um 86 to 100 (the omitted sequences eliminate the genes Ela and Elb and a portion of the E3 gene). From the above information it is to be expected * that whoever is skilled in the art can construct hybrid vectors and viruses containing more or less of the adenovirus gene sequence. Portions of the adenovirus genome in the hybrid virus allow high titers of virus production to occur, often of more than 1 x 10 * 3 plaque forming units (pfu) / rnl. This contrasts sharply with the low titers (1 x 106 pfu / l) that have been found for the recombinant Vñfl.

B. VECTOR COMPONENTS OF VECTOR AND VIRUS The sequences of an adeno-associated virus (Vfifi) are also part of the hybrid vectors and viruses of this invention. The Vfifi sequences useful in the hybrid vector are viral sequences from which the rep and cap sequences encoding polypeptides are omitted. More specifically, the Vfifi sequences employed < *, on the inverted terminal repeat (RTI) sequences 5 'and 3' acting on cis Cvease, for example, B. 3. Crankcase in Handbook of Parvoviruses, ed. P. Ti sser, CRC Press, pages 155-168 (1990) 7. As noted above, the RTI sequences are approximately 143 bp in length. Substantially all the sequences that code RTIs are used in vectors, although it is expected that a certain degree of minor modification * of those sequences is permissible for this use. See, for example, UO 93/24641, published on December 9, 1993. The ability to modify those RTI sequences is within the ability of technicians in this field. For suitable techniques, see, for example, texts such as Sarnbrook and co-authors, Molecular Clomng. fi Laboratory Manual, 2a. edition, Cold Spring Harbor Laboratory, New York (1989). It is possible to obtain the RTI sequences from Vñfi from any known Vñfi, including the types of human Vflfi currently identified. Similarly, Vññ that are known to infect other animals can also be used in the vector constructions of this invention. It is not anticipated that the selection of the Vfifi will mean a limitation for the next invention. A variety of Vfiñ strains, types 1-4, are available from the Fimepcan Type Culture Collection, or can be obtained upon request from a variety of commercial and institutional sources. In the following exemplary mode, a Vfifi-2 is used for convenience. In the hybrid vector construct, the Vfifi sequences are flanked by selected adenovirus sequences, discussed above. The 5 'and 3' Vfifl RTI sequences themselves flank a selected transgene sequence and the associated regulatory elements, described below. Thus, the sequence formed by the transgene and Vfifi sequences 5 'and 3' flanking, can be inserted * at any omission site in the adenovirus sequences of the vector. For example, the Vflfi sequences are conveniently inserted at the site of the Ela / Elb genes omitted from the adenovirus, ie, after the map unit 1. filternativarnent e, the Vñfi sequences can be inserted in an omission of E3, an omission of E2a, etc., If only the RTI 5 '/ em? aq? e sequences of adenovirus and the RTI 3' sequences are used in the hybrid virus, the Vfifi sequences are inserted among them.

C- THE COMPONENT OF TRflNSGENE OF THE HYBRID VECTOR AND THE VIRUS The transgene sequence of the vector and the recombinant virus is a nucleic acid sequence or reverse transcription thereof, hetero logical with respect to the adenovirus sequence, which encodes a polypeptide or a protein of interest. The transgene is operatively linked to the regulatory components in such a way as to allow transcription of the transgene. The composition of the transgene sequence will depend on the use to which the resulting hybrid vector is destined. For example, a type of transgene sequence includes a reporter sequence, which by expression produces a detectable signal. Such reporter sequences include, without limitation, cflDN of beta-galactosidase (LacZ) from E. coll, an alkaline phosphatase gene and a green fluorescent protein gene. These sequences, when associated with regulatory elements that distort their expression, give signals detectable by conventional means, eg, ultraviolet wavelength absorbance, visible color change, etc. Another type of transgene sequence includes a therapeutic gene that expresses a desired gene product in a host cell. These therapeutic genes or nucleic acid sequences typically encode products for administration and expression in an in vivo or ex vivo patient., to replace or correct an inherited or non-inherited genetic defect, or to treat an epigenetic disorder or disease. Said therapeutic genes, which are convenient to implement gene therapy, include, wit limitation, a normal cystic fibrosis transmembrane regulatory (TKGR) gene, a high density lipoprotein (LDL) gene, and several genes which can be easily selected by experts in the field. The selection of the transgene is not considered a limitation of this invention, since said selection is within the knowledge of those skilled in the art. D.- HYBRID VECTOR REGULATOR ELEMENTS In addition to the main elements identified above for the hybrid vector, ie, the adenovirus sequences, the Vññ sequences and the transgene, the vector also includes conventional regulatory elements necessary to force the expression of the transgene in a cell transfected with the hybrid vector, flsí, the vector- contains a selected promoter that is linked to the transgene and located, with the transgene, between the Vfifi RTI sequences of the vector. The selection of the promoter is a routine matter and is not a limitation of the hybrid vector itself. Useful promoters can be * constitutive promoters or regulated (inducible) promoters, which will allow control of the amount of the transgene to be expressed. For example, a desirable promoter is that of the early promoter / blocker, immediate cytomegalovirus Cvease, eg, Boehart and co-authors, Cell, 41: 521-530 (1985) 1. Other desirable promoters include, without limitation, the LTR promoter / promoter of Rous sarcoma virus and the chicken beta-actma promoter. Other promoter / enhancer sequences may be selected by those skilled in the art.

? ? The vectors will also conveniently contain nucleic acid sequences, heterologous with respect to the adenovirus sequences, which include sequences that provide the signals required for efficient pol ladenylation of transcription and codons, with donor sites and receptors. of functional splice. A sequence? Ol? -fl cornun, which is employed in the exemplary vectors of the rese and invention is the derivative of the SV-40 of papovavirus. The poly-fi sequence is generally inserted into the vector after the transgene sequences and before the 3 'RTT sequence. A common intron sequence is also derived from SV-40 and is referred to as the mtron sequence SV-40 T. A vector-hybrid of the present invention may also contain said mtron conveniently located between the enhancer / promoter sequence and the transgene. . The selection of these and other common vector elements are conventional and many such sequences are available Cvease, eg, Sambrook and co-authors, and the references cited therein]. The fiDN sequences encoding said regulatory regions are provided in the plasmid sequence of Figure 2 (SEQ ID NO: 1). The combination of the transgene, the promoter / enhancer, the other regulatory vector elements and the 5 'and 3' Vfifi flanking RTIs are referred to herein as "the rninigene" for ease of reference thereto. As noted previously, the minigene is located at the site of any omission of the selected adenovirus, in the hybrid virus. The size of this enzyme depends on the number and number of omissions in the adenovirus sequence referred to above. Said migene can have a size of 8 kb in the exemplary virus omitted in the El and E3 genes, which is described in the examples that come after, alternatively, if only minimal sequences of adenovirus are used in the virus, this minigene it can have a size of up to about 30 kb. Thus, this hybrid vector and the vector allow a greater degree of latitude in the selection of the various components of the gene, particularly the transgene, with respect to size. Provided with the teachings of this invention, the design of said generator can be carried out using conventional techniques.

E.- THE ASSEMBLY OF THE HYBRID VECTOR AND Lñ PRODUCTION OF THE HYBRID VIRUS The material from which the sequences used in the hybrid vector are derived, the helper viruses, if necessary, and the recombinant hybrid virus (or viral particle) and the various vector components and the various sequences used in the construction of the hybrid vectors of this invention, are obtained from commercial or academic sources, based on previously published and described materials. These materials can also be obtained from an individual patient or generated and selected using techniques from? k molecular cloning reco binantes, common and current, known and put into practice by those who are experts in the field. Any modification of existing nucleic acid sequences, which form vectors and viruses, including sequence omissions, insertions and other mutations, are also generated using common and current techniques. The assembly of the selected fiDN sequences of the adenovirus, the Vfifi and the reporter genes or therapeutic genes and other vector elements, within the hybrid vector, and the use of the hybrid vector to produce a hybrid virus, use conventional techniques, such as those described in example 1. Said techniques include conventional cloning techniques of cfiDN, such as those described in texts (Sambrook and co-authors, cited above), the use of overlapping oligonucleotide sequences, of adenoviruses and Vflfi genomes, the reaction in polymer chain and any suitable method that provides the desired sequence of nucleotides. Common and current transfection and co-transfection techniques are employed, for example, transfection techniques with CaPO.-,, using the human embryonic kidney cell line (REH) 293 of co-plementation (a line of human kidney cells that contains the Ela gene of functional adenovirus, which provides a protein Ela of transaction). Other conventional methods employed in this invention include the homologous recombination of viral genomes, the formation of virus plaques on the agar, methods for measuring signal generation and the like. As described in detail in Example 1 which follows and with reference to Figure 1, a single hybrid virus of this invention is prepared which contains an adenovirus sequence with omitted and partially omitted E3, associated with a single copy of a Vflñ recomb? Nan * te that has omissions of its rep and cap genes, and q? E encodes a selected transgene reporter. Briefly, this exemplary hybrid virus was designed in such a way that the sequence fiV.CMVLacZ (SEQ ID NO: 1) (a mgene containing an RTI of 'Vfifi, a CMV promoter, an SV-40 intron, a LacZ transgene, a pol-~~ SV-40 sequence and a 3'Vfift RTI) were placed in place of the Ela / Elb-type genes 5 (fidd) of adenovirus, which makes adenovirus vector reproduction defective. Due to the limited amount of the adenovir sequence present in the hybrid vectors of this invention, including the pfiV.CMVLacZ (SEQ ID NO: 1) above, a packaging cell line or an auxiliary adenovirus or both, to provide sufficient sequences of the adenovir's gene, necessary for a productive viral infection to generate the hybrid virus. The helper viruses, useful in this invention, contain sequences selected from adenovirus gene, not present in a hybrid vector construct, nor expressed by the cell line in which the hybrid vector is transfected. Optionally, said helper virus may contain a second reporter rninigene that allows the separation of the resulting hybrid virus and the helper virus, when it is purified. The construction of desirable auxilliary viruses is well within the knowledge of those skilled in the art. As an example, if the cell line used to produce the recombinant virus is not a line of packaging cells, and the hybrid vector contains only the minimal adenovirus sequences identified above, the helper virus may be a fid virus of type. wild. In such a manner, the helper virus supplies the required El, E2a, E4 genes of adenoviruses, and all the remaining late, intermediate, structural and non-structural genes of the adenovirus genome. However, if, in this situation, the packaging cell line is 293, which supplies the El proteins, the auxiliary virus does not necessarily contain the El gene. In another embodiment, when the hybrid construct becomes defective in reproduction by a functional omission in El, but does not contain other omissions in the fid genes necessary for the production of an ineffective viral particle, and the 293 cell line is used, a auxiliary virus for the production of the hybrid virus. Finally, all or a portion of the delayed adenovirus E3 early gene (ranging from um 76.6 to 86.2) can be deleted from the helper virus useful in this invention, because this gene product is not necessary for the formation of a hybrid virus particle that works. It should be noted that whoever is an expert in the field can design other auxiliary viruses or develop other emp >cell lines.; aque to complete the omissions of adenovirus in the construction of the vector and allow the production of the hybrid virus particle, given this information. Consequently, this invention is not limited by * the use or description of any particular auxiliary virus or any particular packaging cell line. In such a manner, as described in FIGS. 1 to IC, the plasmid is circular pñd.fíV.CMVLacZ (SEQ ID NO: 1) (which contains the minigene and uniquely adenovirus sequences of the map unit 0 to 1 and from 9 to 16) was digested and transfected with a selected Rd5 auxin virus (containing the adenovirus sequences 9 to 78.4 and 86 to 100), in 293 cells. In that way, the packaging cell line provides the El protein and the helper virus provides all the necessary adenovirus gene sequences, subsequent to the map unit 16. Homologous recombination occurs between the helper virus and the hybrid vector, which results in the hybrid viral particle. The development of this hybrid viral particle in 293 cells has been monitored intimately by more than 20 rounds of amplification, without indication of instability 0 - of the hybrid virus in a host cell and the subsequent modifications of the vector are described below. The hybrid virus ñd.ñV.C l resulting acZ combines the high titre potential of the adenovirus with the integrative biology asoc Lada ! * with the latenc a of VRñ.

G. POLYCHYTHENIC CONJUGATES OF HYBRID VIRUSES The expression of R is necessary for the 0 rescue of the geno to rVfifi to occur. A preferred approach is to incorporate a specific plasmid that allows for the expression of rop within the hybrid particle. In order to do so, the hybrid viruses described above have to be modified further, using the technology of 5 adenov rus-polysine conjugates. See, for example, Uu and coauthors, 3.

Biol. O., 264: 16985-16987 (1989); and K. 3. Fisher and 3. M. Uilson, Biochem .. 3., 299: 49 (April 1, 1994), hereby incorporated herein by reference. By using this technology, a hybrid virus is modified as described by arpba, by adding an ionic polycation sequence, distributed around the capsid of the hybrid viral particle. Preferably the poly cation is polylysine, which binds around the negatively charged virus to form an external positive charge. Then a plasmid or containing the rep gene of Vfiñ (or a functional portion of the same) under the control of a suitable promoter, to the capsid itself, is directly formed under the control of a suitable promoter, to the hybrid oapside, which results in a single vLral particle containing the hybrid virus and a rep gene of Vñfl. The negatively charged plasmid fiDN binds with high affinity to the positively charged polylysine. Essentially the techniques employed to construct this conjugate of hybrid virus or trans-infection particle are described in detail in the following example 3. An alternative embodiment of the hybrid vector and the resulting viral particle is provided by altering the plasmid containing rep so that it also contains a V fi ffi gene cap.

This embodiment of the hybrid vector, when in a host cell, is then capable of producing a recombinant Vfifi pair, as discussed in more detail below. The plasmids used in these embodiments contain conventional plasmid sequences that place a selected VRfl sequence, ie, the rep and / or cap gene sequences, under the control of a selected promoter. In the example provided below, the exemplary plasmid is pRep78 / 52 (SEQ ID NO: 2), a trans-acting plasmid, which contains the Vflfl sequences encoding rep proteins of 78 kD and 52 kD, under the control of the P5 promoter of Vfifl. The plasmid also contains a SV40 polyadenylation signal. The fiDN sequence of this plasmid is provided in Figure 8 (SEQ ID NO: 2).

In a similar manner, and using the vector and vector sequences known in the art, analogous plasmids can be designed using both rep genes, cap genes and different constitutive or regulated promoters, different optional polyfill sequences. The availability of the materials to form those hybrid vectors and modified viruses and the vectors containing Vfifi's rep and / or cap and the techniques involved in the assembly of the hybrid vector and the plasmids that contain rep and / or cap, they are conventional, as described above. The assembly techniques for the trans-infection particle employ the techniques described above for the hybrid vector and the techniques of Uu and coauthors and Fisher and co-authors, cited above. The use of this trans-infection particle, including the rescue and integration of the transgene into the host cell is described below.

II.- LÍ FUNCTION OF THE HYBRID VIRUS A.- HYBRID VIRUS INFECTS A DESTINY CELL Once the hybrid virus or the trans-infection particle is constructed as discussed above, is directed to, or collected by, a selected target cell. The selection of the target cell also depends on the use of the hybrid virus, that is, whether the transgene is to be reproduced or not in vitro for the production of a rechargeable Vfifi particle, or ex vivo, for production at a type of desired cell for delivery back to a patient, om vivo, for delivery to a particular type * of cell or tissue. The target cells for this can be any mammalian cell (preferably a human cell). For example, in vivo use, the hybrid virus can be assigned to any type of cell normally infected by adenovirus, depending on the route of administration, that is, it can be directed, without limitation, to neurons, hepatocytes, epithelial cells and similar. The absorption or collection of the hybrid virus by the cell is caused by the infectious capacity contributed to the vector by the adenovirus and the Vflfl sequences.

B.- THE TRANSGENE IS RESCUED Once the hybrid virus or the trans-infection particle is collected by a cell, the transgene flanked by VTI of the parental adenovirus skeleton must be rescued. The rescue of the transgene depends on the supply of the cell infected with a Vfifi rep gene. Therefore, the efficiency of the hybrid virus can be measured in terms of the rescue mediated by rVñfi rep from the parental adenovirus template. The rep genes can be delivered to the hybrid virus by various methods. One modality to provide rep proteins in trans was demonstrated with the exemplary fid hybrid virus. fiV.CMVLacZ, transfecting to the target rnonolayer of cells previously infected with the hybrid vector, a plasmid gone pRep78 / 52 (SEQ ID NO: 2) wrapped in liposome, containing the genes coding for 78 kDa and 52 kDa proteins of the Vfifi rep, under the role of the Pf promoter of Vfifi. The rescue and amplification of a double-stranded Vfifl of double filament and a double-filament Vñfl dimer, each of which contains the LacZ transgene described above, was observed in 293 cells. This is described in detail in Example 2. The production of rep in trans can be modulated by selection of the promoter in the plasmid containing rep.If high values of rep expression are important early for the rescue of the rechargeable Vflfi domain. , a heterologous promoter (which is not an adenovirus or Vflfi) used to force the expression of rep and eliminate the need for proteins can be used, the low expression levels of rep from P5 occurring in the absence of proteins. The adenovirus may be sufficient to initiate the optimal rescue to force the integration of the recombinant VRfi genome in selected use.It is more preferred for in vivo use that the Vñfl gene also be supplied as a virus. One example of this concept of a single particle is the hybrid polycation conjugate virus (see Figure 7). This is achieved in the same way and with respect to the same target cells that were identified further back. The polylysine conjugate of the hybrid virus, on which it was directly complexed with a plasmid which encoded the rep 78 and 52 proteins, combines all the functional components into a single particle structure. In this way, the transfection particle allows the delivery of a single particle to the cell, which is considerably more convenient for therapeutic uses. Similar experiments to demonstrate the rescue of the transgene from the hybrid conjugate trans-infection particle in the 293 cells and in the HeLa cells are detailed in example 4. In another embodiment, the hybrid virus is modified by directly cloning the cflDN of rep in the adenovirus genome portion of the hybrid vector. Because it is known that even the residual levels of rep expression may interfere with the reproduction of the DN of adenovirus, it is anticipated that such rep incorporation into the hybrid vector itself requires the possible mutation of the rep genes to codify only selected domains. , and the use of promoter promoters to regulate the expression rep, as well as the careful placement of the rep genes within the adenovirus sequences of the hybrid vector.

C- TRANSGENE IS INTEGRATED IN THE CHROMOSOME Once unlinked (rescued) from the genome of the hybrid virus, the recombinant Vñfi minigene / t ransgene searches for a site of integration in the human host and is integrated into it, providing stable expression of the accompanying transgene in the host cell. This aspect of the hybrid virus function is important for its use in gene therapy. The rnimgene sequence of Vflfi / t ransgene, rescued from the hybrid virus, obtains state of prova rus in the target cell, that is, the final event in the life cycle of the hybrid (figure 7). To determine if the Vfifi rnimgene rescued from the hybrid virus obtained the provir? S status in a target cell, HeLa cells were infected that did not express El, with the vector conjugate hybdo-polylysine, complexed with pRep78 / 52 ( SEQ ID NO: 2) and underwent steps until there were clear colonies of cells expressing LacZ. A duplicate plate of cells was infected with the same conjugate, but instead of complexing it with the plasmid pRep78 / 52 (SEQ TD NO: 2), it carried an irrelevant plasmid. Cells that received the hybrid particle containing rep produced a greater number of stable LacZ positive colonies than cells infected with the control vector. This indicates multiple rescue and integration events in the cells that expressed rep proteins. Confirmation of the integration is revealed by the characterization of the reclosing Vflfl genome in the cells infected with the hybrid and chromosomal flanking sequences identi fi ers (see example 5).

III.- THE USE OF HYBRID VIRUSES AND VIRAL PARTICLES IN THERAPY WITH GENES The novel hybrid virus and transfection particles of this invention provide efficient vehicles for the transfer of genes for therapy with somatic genes. These hybrid viruses are prepared to contain a therapeutic gene in place of the LacZ transgene reporter illustrated in the exemplary vector. Through the use of hybrid viruses and transfection particles that contain therapeutic transgenes, these transgenes can be delivered to a patient m alive or ex vivo, to provide integration of the desired gene into a target cell. Thus, these hybrid virus and transfection particles can be used to correct deficiencies or genetic defects.

Two examples of the generation of gene transfer vehicles for the treatment of cystic fibrosis and familial hypercholesterolemia are described in examples 6 and 7 below. Whoever is skilled in the art can generate any number of other gene transfer vehicles including a transgene selected for the treatment of other alterations. For example, it is anticipated that particle transferences are par ticularly advantageous in 1 A therapy with ex vivo genes when desired. Transduction and proviral integration in a stem cell, such as in gene therapy directed to the bone marrow. The hybrid viruses and trans-infection particles of the present invention can be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other sterile, isotonic, aqueous or non-aqueous injection solutions, and other sterile, aqueous and non-aqueous suspensions, which are known to be pharmaceutically acceptable carriers and well known to those skilled in the art, may be employed for this purpose. The hybrid viruses and transfection particles of this invention can be administered in amounts sufficient to transfect the desired cells and provide sufficient levels of integration and expression of the selected transgene to give a therapeutic benefit without undue adverse effects or clinically acceptable physiological effects. that can be determined by experts in the medicinal arts. Conventional and pharmaceutically acceptable routes for administration include? ? direct supply to the target organ, tissue or site, as well as the routes of intranasal, intravenous, intramuscular, subcutaneous, mdermic, oral and other parenteral routes of administration. It can be combined, if desired, the administration routes. The dosages of the hybrid virus and / or the transfection particle will depend primarily on such factors as the condition being treated, the selected gene, the age, the weight and the health of the patient, and can thus vary from patients A therapeutically effective human dose of the hybrid viruses or trans-infection particles of the present invention is believed to be in the range of about 20 to 50 ml of saline containing approximate concentrations of 1 × 10 7 to 1 × 101. °? Fp / rnl of hybrid virus of the present invention. A preferred human dose is around 20 ml of saline at the above concentrations. The dose will be adjusted to balance the therapeutic benefit against side effects. The expiry levels of the selected gene can be monitored to determine the selection, adjustment or frequency of dose administration.

IV.- THE PRODUCTION OF ELEVATED EFFICIENCY OF rVAA The hybrid viruses and transfection particles of this invention have another convenient utility in 3B production of large quantities of particles from Vfifi recornbinant.es. Due to the current m all complicated to generate VRR, there is only a limited amount of Vfifi available for use in industrial, medicinal and academic biotechnology procedures. The vectors and viruses of the present invention provide a convenient and efficient method for generating large quantities of r-Vfifi particles. In accordance with this aspect of the invention, a transfection particle is constructed as described above and in Example 3, and is used to produce high levels of rVfifl, as detailed in Example 8, with the possible modifications described in FIG. example 9 that comes later. Briefly, a plasmid is generated that contains both the rep genes and the cap genes of Vfifi ba or the control of a suitable plasmid, and is complexed with the outer polylysine of the hybrid virus, as described above. This transinfection particle is then allowed to infect a selected host cell, for example, 293 cells. The presence of rep and cap allows the formation of Vfifl particles in the cells and generates a Vñfi virus titer of about 1Q9. I saw nones. In contrast, current methods involving the transfection of multiple plasmids only produce a titer of about 10 virions. RVfifl is isolated from the culture by selecting the blue plates containing LacZ and purifying them in a gradient of cesium chloride. The benefit of this procedure refers to the fact that the cis Vfifi element is encoded by the parental adenovirus genome. As a result, the trans plasmid is the only component of ñDN that is necessary for complex formation. In that way the cell is loaded with significantly more copies of the rep and cap sequences that drive in trans, which results in improved rescue and packing efficiency. Numerous comparative studies that focus on the optimal ratio and number of copies of the cis and trans plasmids for Vññ production indicated that there is a positive correlation between the number of trans plasmid copies and the yield of recombinant virus. As described, for example, in detail in Example 8, the performance of the recombinant virus ñV.CMVLacZ was increased by 5-10 times by the use of the transfection particle, instead of a common and current adenovirus vector. . The main limitation associated with the production of Recombinant Vfifl, using a hybrid virus of this invention refers to the difficulties that arise in the distinction between the two viruses (ie the adenovirus and Vflfl), which are produced by the cell. Using the exemplary vectors and exemplary vector components of this invention, the LacZ histochemical stain could not be used to titrate the yield of recirculating fiV-CMVLacZ, since any polluting hybrid fldflV.CMVL acZ would contribute to the final count. Therefore, a rapid Southern spotting technique was incorporated to quantify the yields of recombinant Vflfi. The analysis that was developed allowed not only the codification and verification of the Vflfl production, but also demonstrated the elimination of the hybrid polluting virus with respect to the purified Vfifl materials. Another method for detecting hybrid viruses contaminates is to modify the hybrid vector by inserting a second small inigene reporter (i.e., reporter gene sequences, promoters and other expression control sequences, when desired) into the E3 region of the skeleton of parental adenovirus. Because this reporter is not linked to the Vñfi domain, the contaminating hybrid virus that is present during purification can be monitored by this specific marker for the hybrid. Another possible reporter gene is the nucleic acid sequence for the green fluorescent protein. With this hybrid vector containing two reporter sequences, the histochemical stain activity for alkaline phosphatase (adenovirus reporter) or beta-galactosidase (Vflfl reporter) can be used to monitor each viral domain. The following examples illustrate the construction and testing of the hybrid vectors of the present invention and their use in the production of recombinant Vñn. These examples are illustrative only and do not limit the scope of the present invention.

EXAMPLE 1 CONSTRUCTION OF A HYBRID VIRUS A first hybrid virus of adenovirus-fifi was denoted by homologous recombination between ñDN extracted from an adenovirus and a complementing vector *, according to the previously described protocols Cvease, for example, K. F.

Kozarsky and coauthors, J. Biol. Chem., 269: 13695-13702 (1994) and references cited there !. The following description refers to the diagram of figure 1. The adenovirus fiRNA was extracted from virions dl7001 purified with CsCl, a variant Rd5 (serotypic subgroup C) which leads an omission of 3 kb between? M 78.4 to 86 in the E3 region non-essential (provided by Dr. Uilliarn Uold, Washington Umversity, St. Louis, Missouri). Adenoviral fiDN was prepared for cotransfectacaon by digestion with Clal (adenovirus genomic position of pb 917) which eliminates the left branch of the genome that spans the map units 0-2.5 of adenovirus. See the bottom diagram of figure IB. The complementary vector hybrid, pfid.fiV.CMVLacZ (see figure ifl and figure 2 CSEQ ID NO: 1)) was constructed as follows: A parental cloning vector, pfid.BglII, was designed.

It contains two wild-type ñd5 genome segments (ie, apa units 0-1 and 9-16.1) separated by a unique BglII cloning site for the insertion of heterologous sequences. The missing fid5 sequences between the two domains (p.b. 361-3327) of the adenovirus genome result in the omission of Ela and the majority of Flb, followed by recornbmación with viral fiDN. A recombinant Vññ genome was designed (ñV.CMVLacZ) and inserted into the BglII site of pfld.BglII to generate the complementing plasmid. The linear arrangement of fiV.CMVLacZ (SFQ ID NO: 1) (see the upper diagram of Figure IB) includes: (a) The 5 'VNñ RTI (p.b. 1-173) obtained by PCR, using pVñ2 CC. ñ. La? Ghlin and co-authors, Gene, 23: 65-73 (1983) 1 co or nucleotide template nucleotide 365-538 of Figure 2 (SEQ ID NO: 1) 1; (b) an early promoter / promoter, immediate to CMV CBoshart and co-authors, Cell, 41: 521-530 (1985) 1; n-cleotide numbers 563-1157 of Figure 2 (SEQ ID NO: 1) 1; (c) a splice-receiver splice receiver SV40 Nucleotide numbers 1178-1179 of Figure 2 (SEQ ID NO: 1) 1; (d) cflDN of beta-galactosidase from E. coli Nucleotide nucleotide 1356-4827 of Figure 2 (SEQ ID NO: 1); (e) a SV40 polyadenylation signal (a restriction fragment HI-BclI of 237 Bam, which contains the division / pol-fi signals of both the early and late transcription units, nucleotide numbers 4839-5037 of the Figure 2 (SEQ TD NO: 1) 1; and (f) RTI of 3'VRfi, obtained from pVR2 as a SnaBT-BglII fragment Nucleotide Nos. 5053-5221 of Figure 2 (SEQ ID NO: 1). Complementing hybrid vector, resulting, pfid.fiV.CMLacZ (see Fig. Ifi and Fig. 2 SEQ ID NO: ll), contained a single copy of recombinant ñV.CMVLacZ, flanked by * lae coordinates of adenovirus 0-1 on one side and 9-16.L on the other The plasmid ñDN was linearized using an NheT site immediately 5 'to the zero (0) map unit of adenovirus (which results in the upper diagram of Figure IB). Both the co-adenovirus substrate or the complement vector flDN were transfected into 293 cells (fiTCC CRL1573) using a standard procedure. common transfection technique with calcium phosphate (see, for example, Sambrook and co-authors, cited above). The final result of the homologous recombination involving sequences that map the map units 9-16.1 of adenovirus is the hybrid fid.fiV.CMVLacZ (see figure IC), where the Ela and Elb coding regions of the adenovirus substrate dl7001 they are replaced by the fiV.CMVLacZ of the hybrid vector. Twenty-four holater, the transfectation mixture was removed and the cells were covered with 0.8% agarose containing 1x BME and 2% fetal bovine serum (FBS). Once the viral plaques developed (typically 10-12 days after transfection); The plaques regarding the activity of bota-galac osidase from E. coli (LacZ) were covered by covering the monolayer infected with agarose supplemented with a histochemical stain for LacZ, according to the procedure described in 3. Pnce and co-authors, Proc. Nati, ficad. Sci., USA, 84: 156-160 (1987). Positive clones (identified by the insoluble blue deposit) were isolated, subjected to three rounds of freezing (dry ice / ethanol), defrosting (37 ° C) and an aliquot of the suspended plate was used to infect a monolayer. fresh from 293 cells, seeded on duplicate plates of 60 m. Twenty-four holater, the cells of a series of plates were fixed and again stained for the activity of LacZ. The cells of the duplicated plate were harvested, suspended in 0.5 nl of 10 inM Tps-Cl, pH 8.0, and subjected to lysis, carrying out a series of three freezing cycles (dry ice / ethanol) - thawing ( 37 ° C). The cell debris was removed by centrifugation and an aliquot of the supernatant was used to measure the enzymatic activity of L cZ. As indicated in FIG. 3, the analyzes for beta-galactosidase activity, which measured the absorbance at 420 nm of the blue color of beta-galactosidase in satisfactory recornbi nants, revealed that eight of the ten positive putative clones isolated (ülfi to D1J) expressed high levels of enzyme. Histochemical staining yielded similar results. The production and large scale purification of recirculating virus was carried out as described by Kozarsky and coauthors, cited above, and in the references cited by them.

EXAMPLE 2 FUNCTIONAL ANALYSIS OF THE HYBRID VECTOR The ability to rescue the sequence flv.CMVLacZ (SEQ ID NO: 1) of the hybrid virus, represented an important aspect of the hybrid vector and virus systems of example l. To assess this aspect, it was necessary to provide the necessary Vñfi gene products in trans, which would direct the excision of Vfiñ and its amplification (that is, the rep proteins). Finally, this experiment was carried out on 293 cells to transfect the omission in the fid clones. fiV.CMVLacZ, because the adenovirus gene proteins have proved important in initiating the lithic phase of the Vfifi life cycle. 293 cells were plated in 35-m plates, with 6 concavities, at a density of 1 x 106 cells / concavity.

Twenty-four hours later replacing the CDMEM / 10% FBS seed medium, supplemented with antibiotics! with 1.0 ml of DMEM / 2% FBS and was infected with hybrid clones fidñV.CMVLacZ, at a MOI of 1. Two hours later each concavity was transfected with 1 μg of plasmid gone pRep78 / 52 (SEQ ID NO: 2), a plas transaction encoding the sequence encoding Vñfi rep proteins of 78 kD and 52 kD. The rep sequences in this construction are under the control of the Vfifi P5 promoter and use the SV40 polyamide signal. As a positive control for the rescue of Vññ, 293 cells were seeded in a plate of 6 concavities, as above, and transfected with a plasmid of Vfifi that acts in cis, pfiV.CMVLacZ and pRep78 / 52. pFíV.CMVLacZ contained flV.CMVLacZ, the identical sequence encoded by pñd.ñV.CMVLacZ (SEQ ID NO: 1) described in example 1, cloned in the BglII site of pSP72 (Promega). To provide the adenovirus helper function necessary for the rescue of Vññ, the cells were infected with a wild type fid5 virus or with a fid.CMhpfiP virus, with omission in El, of first generation, to an MOT of 5, approximately two hours before adding the transfectation mixture. The fid.CMhpfiP is identical to fid.CMVLacZ (example 1), with the modification that the alkaline phosphatase sequence (which can be obtained from Genbank.) Is inserted instead of the LacZ gene. Transfectation was carried out with Lipofectarnine (Life Technologies) according to the instructions provided by the manufacturer. Thirty hours after the transfection, the cells were harvested and the episomal fiDN was prepared (Hirt extract), as described by 3. M. Uilson and co-authors, 3. Biol. Chern., 26? : (16): 11483-11489 (1992). The samples were resolved on a 1.2% agarose gel and electornorncho on a nylon membrane. The spots (Southern) were hybridized with a restriction fragment randomly labeled with a 32P sensitizer isolated from the LacZ cflDN of E. coll. The full spectrum of duplex molecular species that appeared during an infection with lytic Vfifi (ie, monornépcas forms of double-stranded intermediates, RF and RFd, respectively), was evident in the transfected cells, infected with the wild-type and ñd5 with omission of El. Intermediation of reproduction was not detected when transfections were carried out in the absence of the auxiliary virus. Hirt extracts from the 293 cells infected with putative fiV.CMVLacZ Rd.D.D. and D1C hybrid clones revealed a single band corresponding to the viral RDN when probed with a LacZ restriction fragment. However, in the presence of the re? 78 and 52 proteins, the same clones produced a band pattern that included not only the adenovir? RDN, but also an RF monomer and a fiV.CMVLacZ dimer. It was not evident in the form of a single filament of fiV.CMVLacZ (SEQ ID NO: l). Two additional clones gave similar band patterns, DIB and DIH. In all, each of the eight fid.fiV.CMVLacZ hybrids that were found in Figure 3 that expressed high levels of LacZ activity were positive for the rescue of the VRR domain. With the exception of an extra band of approximately 3.5 kb, the rescue of the fiV.CMVLacZ (SEQ ID NO: 1) of the viral hybrid ñDN was almost identical to the results obtained from * an approach based on the plasma standard L cis and trans. In these latter samples, the auxiliary function of adenovirus was provided by cells previously infected with wild ñd5 je t po or with a recombinant virus fld.CBhpñP with Fl oimsión (also called Hd.CBñLP). The fld.CBhpñP virus has the same sequence as the ñd.CMhpfiP virus described above, except that the CMV promoter sequence is replaced by the chicken cytoplasmic beta-actin promoter Cnucleotides +1 to +275, which are described in T. ñ. Kost and co-authors, Nucí. flcids Res., 11 (23) r.8287 (1983) 1. The level of rescue in the cells infected with UT 0d5 seemed to be higher with respect to those infected with the recombinant fid.CBhpflP virus, probably due to the additional expression of El, provided by the wild-type genome. The relevance of including an adenovirus with omission here is to document that the level of Adenovirus proteins, expressed in 293 cells, is sufficient for the function of Vfifl helper.

EXAMPLE 3 SYNTHESIS OF POLYLYSINE CONJUGATES Another version of the viral particle of this invention is a polylysine conjugate with a rep plasmid formed complexed directly with the hybrid virus capsid. This conjugate allows the efficient delivery of the expression plasmid rep pRep78 / 52 (SEQ ID NO: 2) in tandem with the hybrid virus, thereby eliminating the need for a separate transfection step. See Figure 8 for a diagramatic trace of this construction. Purified materials were modified from a large scale expansion of the Dlfi clone of ñd.ñV.CMVLacZ, coupling? Ol? -L-lysine to the vipon capsid, essentially as described by K. J. Fieher and 3. M. Uilson, Biochem. 3., 299: 49-58 (1994), which resulted in a conjugate fld. flV. CMVLacZ - (Lys) n- The procedure involves three steps. First the hybrid virions are activated by primary amines on capsid proteins, with the water-soluble heterobi functional crosslinking agent, sulfo-SMCC Cl-carboxa Sulfo- (N-succ? Nim? D? L) -4- ( N-rnale? M? Dome il) -cyclohexanol (Pierce) The conjugation reaction, which contained 0.5 rng (375 nmol) of sulfo-SMCC and 6 x 1012 pairs of the hybrid vector A260 in 3.0 i of HBS, was incubated at 30 ° C for 45 minutes with constant gentle shaking. This step comprised the formation of a peptide ligature between the active Nh? D? -ox? Succ? N? M? Da (NHS) ester of s? Lfo-SMCC and a free amine (eg, lysine), provided by the protein sequence of adenovir? s (capsid protein) in the recombinant virus, which produces a viral particle activated with maleirnide. The unreacted sn reactive interlayer was removed by gel filtration on a 1 cx 15 cn column of Bio-Gel P-6DG (Bio-Rad Laboratories), equilibrated with 50 mM Tps / HCl buffer, pH 7.0 and 150 rnM NaCl. The fractions with ñ2β peak that contain the hybrid virus activated with maleirnide, were combined and placed on ice. Secondly, the pol-L-lysine having a molecular mass- from 58 kDa to 10 mg / ml in 50 rnM of regulator tpetanolarnina (pH 8.0), 150 rnM of NaCl and 1 rnM of EDTA, with 2 rnM, was pulled out. -hyinothiolane / HCl (Reagent from Trau, Pierco) at a rnolar ratio of 2 moles of SH / mol of polylysine, under 2; the cyclic thio-nitidate reacts with the primary amines of pol (L-lysine), which results in a thiolated polycation. After a 45 minute incubation at room temperature, the reaction was applied to a 1 cm x 15 cm column of Bio-Gel P6DG, equilibrated with 40 M Tps / HCl buffer (pH 7.0, 150 mM NaCl and 2 mM EDTñ to eliminate the unincorporated Traut reagent The quantification of free thiol groups was achieved with Ellman's reagent [5, 5'-d?? ob? s- (2-nitrobenzoic acid) 1, which reveals approximately 2 moles of SH / rnol of? ol? (Ll? s? na) The coupling reaction was initiated by adding lx 1012 particles of R260 of the hybrid virus activated with male ida / rng of pol (L-lisma t olada, e The mixture was added on ice at 4 ° C for 15 hours, under argon.Radcaptoethylamine was added at the completion of the reaction and the incubation was carried out at room temperature for 20 minutes to block the unreacted allene sites. The conjugates of vi rus- ol limna, ñd.ñV.CMVLacZ- (Lys) n with respect to the pol i (L - 1 ísi na) not conjugate were purified. This was achieved by ultracentrifugation through a step gradient of CsCl with an initial composition of equal volumes of 1.45 g / rn (lower step) and 1.2 g / ml (upper step) of CsCl in 10 mM of regulator-Tris / HCl ( pH 8.0). Centrifugation is carried out at 90,000 g for twelve hours at 5 ° C. The final product was dialyzed against 20 nm Hepes regulator (pH 7.8) containing 150 nM NaCl (HBS). Ad.AV.CMVLacZ- (Lys) n complexes were formed with DNA from plasmid pRep78 / 52 (SEQ ID NO: 2) by adding various amounts of fid.OV.CMVLacZ- (Lys) n in 50 μl of HBs at 0.5 μg of ñDN of plasmid pRep78 / 52 (SEQ ID NO: 2) in 50 μl of HBs. After 30 minutes of incubation at room temperature, a complex of hybrid virus fld.fíV was formed. CMVLacZ- (Lys) n, associated in a single particle with the plasmid DNA containing the r p genes. This complex, called a particle of trans- '•, *. "" Infection was assessed in terms of its ability to bind to RDN by means of gel mobility displacement analyzes, carried out as described by Fisher and coauthors, cited above. This analysis revealed that the plasmid-binding capacity of the purified conjugate (expressed as the number of particles f ß or of fid.fiV-CMVL cZ- (Lys) n that can neutralize the charge contributed by 1 μg of plasmid fiDN) was 1 μg of fiDN of the plasmid gone? Re? 78/52 / 6.0 x 10 ^ 0 fi2o particles of Ad .RV. CMVLacZ- (Lys) n.

EXAMPLE 4 TRAN5INFECTION PROTOCOL TO DEMONSTRATE VAA SEPARATION AND AMPLIFICATION Transfection complexes were prepared by mixing the fid conjugate. RV.CMVLacZ- (Lys) n with the plasmid pRep7R / 52 (SEQ ID NO: 2) and was applied to 293 cells in the same way. Fid.fiV.CMVLacZ- (Lys) "(5 x 10 particles of fi2ß) in 100 μl of DMEM was added dropwise to a microfuge tube containing 1 μg tle ñDN of plasmid in 100 μl of DMEM. The mixture was mixed gently and allowed to incubate at room temperature for 10-15 minutes. The transfection mixture was added to 293 cells seeded in a plate of six concavities, of 35 nm, as detailed above. Thirty hours later the cells were harvested and Hirt extracts prepared. Samples were solved in a 1.2% agarose gel and electrophoresed on a nyLon membrane. The blots (Southern) were hybridized with a restriction ment labeled with randomizer P-32, isolated from LacZ cflDN of E. coli. Hirt extracts of 293 cells revealed a pattern of bands that suggested the sequence of my igene ñV. CMVLacZ (SEQ ID NO: 1) was efficiently rescued from the hybrid conjugate. Both the RF monomer and the dimer of the sequence FV.CMVLacZ recorn i nan e were evident. As was previously observed, the rescue event was dependent on the rep proteins, since 293 cells that were transinfected with a hybrid conjugate, formed a complex with a plasmid reporter irrelevant, that expressed alkaline phosphatase (ie, pCMVhpfiP) only revealed DNA from ñd. fiV.CMVLacZ. This negative control for the rescue was secondarily useful to demonstrate the high efficiency of the gene transfer to 293 cells that was obtained with the conjugate vehicle. A duplicate series of 293 cells that received the hybrid conjugate, which had been additionally formed to complex with the alkaline phosphatase expression plasmid, was fixed 24 hours after the addition of the transfection mixture and stained histochemically for LacZ, such as was described by Pnce and coauthors, cited above, or for alkaline phosphatase activity, as described by * 3. H. Schreiver and coauthors, BioTechniques, 14: 818-823 (1993). flqui, LacZ was a marker for the Ad hybrid. AV. CMVL acZ, while the alkaline phosphatase served as an informer for the plasmid carrier. More than 90% of the monolayer was transduced with both the beta-galactosi dasa transgenes and the alkaline phosphatase transgenes, which showed the evadability of the conjugate delivery vehicle (the differential spotting reveals a color blue for the hybrids that continue with the LacZ marker and a purple color for the plasmids carrying the RP marker). Due to the important role that the proteins have for the advance in the life cycle of Vfifi, it was critical to prove * the efficiency of the hybrid supply system in a setting where the El proteins were not expressed. Therefore, it was performed a transinfection experiment using the hybrid conjugate formed complexed with pRep78 / 52 (SEQ ID NO: 2) in HeLa cells (ATCC CCL2) to eliminate the involvement of the El proteins. The findings suggested that the rescue of fiV.CMVLacZ occurred. evidenced by the accumulation of RF monomers and conductors. The rescue of HeLa cells (which, unlike 293 cells did not contain any Adenovirus protein) revealed lower levels of transgene rescue. The expression of rep from the P5 promoter of AAV is upregulated by the adenovirus and signals the beginning of the lytic cycle of the AAV. In the absence of El, the expression of rep from the P5 promoter is virtually silent, which is important for the maintenance of the latent stages provi rales of the Vflfi life cycle. It is anticipated that a promoter not dependent on the expression of El would solve this problem by substituting P5.

EXAMPLE 5 THE INTEGRATION OF TRANSGENE A preliminary study had been made to determine if the Vfifi sequence rescued from the hybrid virus could obtain a provirus status in a target cell (Figure 7). Briefly, HeLa cells (RTCC CCL 2) were infected with the hybrid conjugate, complexed with? Rep78 / 52 (SEQ ID NO: 2) and subjected to steps until stable colonies of cells expressing LacZ were evident. A duplicate plate of cells was infected with the same conjugate, but instead of being complexed with the plasmid pRepi'8 / 52 (SEQ ID NO: 2) it carried an irrelevant plasmid. These findings indicated that the cells that received the hybrid particle containing Rep produced a greater number of stable LacZ positive colonies than the cells that had been infected with the control virus. This could be interpreted as a reflection of multiple salvage and integration events in cells expressing Rep proteins. However, it is possible that an episodic form of AAV that may persist for prolonged periods was present.

To establish the occurrence of integration in the chromosome of the min gene Rv. CMVLacZ from the hybrid conjugate the following experiment was carried out. The conjugate Rd.AV.CMVLacZ- (Lys) n, which carries the pPLep78 / 42 plan (SEQ ID NO: 2) was used to infect HeLa cells (RTCC CRL2) (primary fibroblasts can also be used). The infected cells are subjected to steps (for several generations), the cells develop to confluence, divide and let them grow again until confluence, divide again and repeat the cycle as many times as desired.This allows a sufficient time for capture, expression, reproduction and integration to occur, see Figure 7. To verify that the recombinant Vfifl sequence that was rescued from the hybrid genome (step III of figure 7) has been integrated into a omosome of the host cell (step IV of figure 7), the cells are separated in a fluorescence activated cell sorter (FRCS) .Through this technique, those cells that contain an integrated, stable copy of the recombinant igene ñV.CMVLacZ are separated, based on the presence of the beta-galactos dasa reporter.These cells are labeled with fluorescein-labeled antibodies, which recognize the ß-Gal protein and then separate them from the s cells not transduced, that is, those that did not receive a copy of the rninigene Vñfl), by FfiCS. The DNA is isolated from this purified population of cells and used to construct a genomic bank that is discriminated for individual clones and the sequence is verified. If integration occurred, it is documented directly by analysis of the sequence.

EXAMPLE 6 THE GENE TRANSFER VEHICLE FOR CISTIC FIBROSIS An adenovir? S-Vfifi-RTFC virus was constructed by modifying the hybrid virus fid.RV .CMVlacZ described in example 1, to contain the cystic fibrosis transrnembrane regulatory gene (RTFC) CJ. R. Riordan and coauthors, Science, 245: 1066-1073 (1989) 1 instead of the lacZ gene, using known techniques. A suitable method involves producing a new vector using the techniques described in example 1. In this new vector the ninigene LacZ is replaced by the minigene RTFC. For the performance of this method, the vectors that carry the RTFC gene have been previously described and can be easily constructed. This new or reconstructed vector is used to generate a new virus by means of homologous recombination, as described above. The resulting hybrid virus is called the hybrid fld.flV.CMVRTFC. It has the sequence of figure 2 (SEQ ID NO: 1), except that the LacZ gene is replaced by RTFC. Alternatively, the LacZ gene of the vector ñd.ñV.CMVLacZ of Example 1 can be deleted and replaced by the RTFC gene, using known techniques. This virus (or an analogous hybrid virus), with a different promoter, regulatory regions, etc. different) is useful in therapy with genes alone or, preferably, in the conjugated conjugate form as described in example 4. The treatment of fibrocysticity, using the viruses provided above, is particularly suitable for therapy with genes in vivo, directed to the lung. The airway epithelial cells are the targets or most convenient destinations for gene transfer, because the pulmonary complications of CF usually mean more morbid and more life-limiting expression. Thus, the vector-hybrid of this invention, which contains the RTFC gene, is delivered directly to the respiratory tract, for example, by formulating the above hybrid virus in a preparation that can be inhaled. For example, the hybrid or conjugated virus of the invention, which contains the RTFC gene, is suspended in 0.25 molar sodium chloride. The virus or conjugate is absorbed by the airway cells and the gene is expressed. Alternatively, the hybrid viruses or conjugates of the invention can be delivered by other suitable means, including site-directed injection of the virus containing the RTFC gene. In the case of the supply of the RTFC gene, the preferred solutions for bronchial instillation are sterile saline solutions containing the recombinant hybrid virus of the present invention in an approximate scale of 1 × 10 7 to 1 × 10 10 pfu / ml, more particularly, within from the approximate scale of 1 x 108 to 1 x 109 pfu / rnl. Other suitable methods for the treatment of cystic fibrosis, by the use of recombinant viruses for gene therapy, of this invention, can be obtained from discussions in the art of other types of gene therapy vehicles for FC. See, for example, U.S. Patent No. 5,240,846, incorporated herein by reference.

EXAMPLE 7 THE GENE TRANSFER VEHICLE FOR FAMILY HIPPERCOLESTERQLEMIA Familial hypercholesterolemia (FH) is an autosomal dominant disorder, caused by abnormalities (deficiencies) in the function or expression of CM LDL receptors. S. Brown and J. L. Goldstein, Science, 232 (4746): 34-37 (1986); J. L. Goldstein and M. S. Brown, Familial hypercholesterolemia in Metabolic Basis of Inhepted Disease, ed. C. R. Scríver and co-authors, McGraw Hill, New York, pages 1215-1250 (1989) 1. Patients who inherit an abnormal allele have moderate elevations of LDL in the plasma and suffer from premature diseases in the coronary arteries (EñC), which threaten their life. Homozygous patients have 6 (1 severe hypercholesterolernia and pJflC that threatens s? gone in childhood. The adenovirus-Vfiñ-LDL hybrid virus of the invention is constructed by replacing the lacZ gene in the hybrid virus ñd.fiV.CMVlacZ of Example 1 with an LDL TT receptor gene. Yamamoto et al., Cell, 39: 27-38 (1984) 1 using known techniques and as described analogously to FC in the preceding example. The vectors carrying the LDL receptor gene according to this invention can be easily constructed. The resulting hybrid vector is called pfid.fiV.CMVLDL. This plasmid or its recirculating virus is useful in therapy with HF genes alone or, preferably, in the form of a viral conjugate, prepared as described in Example 4, to replace a normal LDL gene with a Abnormal allele, responsible for the gene.

A. THERAPY WITH EX VIVO GENES Therapy with ex vivo genes can be carried out by harvesting and establishing a primary culture of hepatocytes from a patient. Known techniques can be used to isolate and transduce the hepatocytes with the vector * or the previous vectors carrying the gene or the LDL receptor genes. For example, collagenase perfusion techniques, developed for rabbit liver, can be adapted for human tissue and can be used in transduction. After transduction, the hepatocytes are harvested from the tissue culture plates and reinfused into the patient, using known techniques, for example, by means of a catheter placed in the inferior mesenteric vein.

B.- THERAPY WITH IN VIVO GENES Conveniently, the in vivo approach to gene therapy, for example, directed to the liver, involves the use of the hybrid viruses and the viral conjugates described above. A preferred treatment comprises infusing a trans-infection particle of the invention which contains LDL into the peripheral circulation of the patient. The patient is then evaluated for the change in serum lipids and liver tissues. Hybrid virus or a viral conjugate can be used to infect hepatocytes in vivo, by direct injection into a peripheral or portal vein (10? -108 pfu / kg) or back into the biliary tract (same dose). This effects gene transfer in most hepatocytes. The treatments are repeated as necessary, for example, weekly. It is anticipated that administration of a dose of virus equivalent to an MOI of around 20 (ie, 20 pfu / hepatocyte) will lead to high level gene expression in most hepatocytes.

EXAMPLE 8 EFFICIENT PRODUCTION OF RECOMBINANT VAA USING A VIRUS HYBRID / CONJUGATE The following experiment showed that the genome of Vftfl that was rescued from the hybrid virus Rd.fiV.CMVLacZ could be packaged in a VRR capsid, as long as the cap open reading frame is supplied in the trans position. Thus, the viruses of this invention are useful in a production method for recombinant VRR, which solves the complications of the prior art that surround the high titre production of recirculated VRR.

A. THE TRANSFER PROTOCOL FOR THE PRODUCTION OF RVAA A transfection complex was formed, composed of the conjugate Ad.AV.CMVLacZ- (Lys) n, described above, and a pAdAVAñ transcomplementing plasmid, which is described in detail in R. J. Samulski and co-authors, J. Virol. , 63 (9): 3822-3828 (1989). Briefly, the pñdVñfl plasmid encodes all open reading frames rep and cap in the absence of The Vññ RTIs have been shown to provide the necessary Vfifi auxiliary functions for the reproduction and packaging of the recombinant Vññ sequences. The conjugate fid.fiV is added dropwise. CMVLacZ- (Lys) i (4.5 x 1013 R26O particles) in 75 ml of DMEM, with moderate constant circular stirring in 20 ml of DMEM, containing 750 μg of the pfidVfifi helper plasmid and incubated at room temperature for 10-15 minutes. The complex was diluted with 450 ml of DMEM, supplemented with 2% FBS and 20 nl aliquots were added to monolayers of 293 cells seeded on 150 nm plates. Forty hours after the transfection, the cells were harvested, suspended in 12 ml of 10 mM Tps-Cl (pH 8.0) and stored at -8 ° C. Because the anticipated result was the production of the hybrid virus ñd.ñV.CMVLacZ and a recirbinant Vñfl virion (AV.CMVLacZ), both of which carry a functional LacZ rnmigene, it was not possible to use the detection of the activity of LacZ co or an indicator of the production of AV.CMVLacZ. A novel molecular approach was developed that could be carried out in one day and allowed the identification of the packed viral ñDN.

B.- Lñ PURIFICATION OF rV A Briefly, the suspensions of frozen cells were subjected to three rounds of freeze / thaw cycles to release the reclosing AV.CMVLacZ and the hybrid fld.fiV.CMVLacZ. When final thawing is complete, bovine pancreatic DNase (2,000 units) and ribonuclease (0.2 mg / ml final concentration) are added and the extract incubated at 37 ° 0 for 30 minutes. The cells were discarded by centrifugation (5,000 xg for 10 minutes) and the clarified supernatant (15 ml) was applied to a step gradient of 22.5 rnL, composed of equal volumes of CsCl at 1.2 g / ml, 1.36 g / ml and 1.45 g / ml of 10 M Tris-Cl, pH 8.0. Bands were formed with the viral particles at 25,000 rprn in a Beckman S -28 rotor for 8 hours at 4 ° C. Fractions of 1 ml were collected from the bottom of the tube. The fractions recovered from the CsCl gradient, of the partially purified virus, are then digested to release the viral ñDN from the virion capsids, in the following manner. A 5.0 μl sample of each fraction was transferred to a microfuge tube containing 20 μl of capsid digestion buffer (50 mM Tris-Cl, pH 8.0, 1.0 mM EDTfl, pH 8.0, 0.5% SDS and 1.0 rng / protein protein K). The reaction was incubated at 50 ° C for one hour, allowed to cool to room temperature, diluted with 10 μl milli-Q water and agarose gel loading dye was added. Then these fractions were analyzed by Southern staining. The samples were resolved on a 1.2% agarose gel, electro-welded onto a nylon membrane. A restriction fructose LacZ labeled with 32 pj was used, which was common to both vectors, as a hybridization probe to localize the migration of the viral DNA through the agarose gel. Viral bands were quantified in a Phosphoirnager imager from Molecular Dynamics.

A sample of the extract was also tested before binding with CsCl, and revealed both fiDN of ñd.ñV.CMVLacZ, as double-filament RF forms (monomers and digesters) of the rescued sequence flV.CMVLacZ (SEQ Tü NO: 1) . A single filament monomer of fiV.CMVLacZ appeared to be present in the crude extract; however, it was not until the vipones were concentrated by ult racent pfugation of floating density, that the single-strand genome was clearly evident. The genome recornbi ante of a single strand of the virus was distributed on a scale of densities of CsCl and rebell or a biphasic band-forming pattern. The two peaks of the single filament rVA genome occurred at densities of 1.41 and 1.45 g / ml CsCl, consistent with the reported floating densities of wild type Vflfl in CsCl CL. M. de la Maza and coauthors, 3. Virol., 33: 1129-1137 (1989) 1. The analysis of the fractions corresponding to the two vector forms * revealed that the rVflñ-1 species. 1 were vain orders of magnitude more active for lacZ transduction than the denser variant of 4VAA-1.45 g / ml. To avoid confusion with the contaminant samples of Ad.VAA, were thermally activated (60 ° C for 30 minutes) before adding them to the HeLa indicator cells. The p co fractions of rVA-1.41 were combined and purified by equilibrium sedimentation in CsCl to remove the residual adenovirus particles and concentrate the rVññ vinons. In the final round of ultracentrifugation, a weak, but clearly visible, opalescent band was observed in the middle of the gradient tube. The fractions surrounding the band were evaluated for density, absorbance at 260 nn and lacZ transducer particles. As the band was eluted from the gradient tube, a well defined peak of absorber material at 260 n was recorded, with maximum absorbance occurring at a density of 1.40 g / rnl of CsCl. The analysis for the lacZ transducer particles in HeLa cells revealed a peak of activity that mirrored the absorbance profile. These results indicate that rVñfi was produced from the hybrid fiD.Vflfi virus. Traditionally, the titers obtained using the hybrid virus were elevated 5 to 10 times compared to the more conventional recombinant VRfi production schemes (ie, transfectations with plasmids acting in cis and trans). This represents a major improvement in the production of rVRfi and indicates that the hybrid is useful for the production of rVfifl on a large scale. All the references mentioned in the foregoing are incorporated herein by reference. Numerous modifications and variations of the present invention are included in the specification identified above, and are expected to be obvious to those skilled in the art. Such modifications and alterations to the compositions and methods of the present invention, such as those modifications that allow optimal use of the hybrid viruses as gene therapy vehicles or production vehicles for the production of recompressive VRO, are believed to they fall within the scope of the claims that read at the end of this.UENCES (1) GENERAL INFORMATION: (i) APPLICANT: Trustees of the University of Pennsylvani. Uil are, JJJJarnes M. Kelley, Uill íarn M. Fisher-, Kpshna 1. (il) TITLE OF LR INVENTION: HYBRID VIRUS OF RDENOVIRUS- FIRE-DESIGNED VIRUS AND METHODS FOR YOUR USE (lil) NUMBER OF SEQUENCES: 2 (IV) ADDRESS PflRfl CORRESPONDENCE: (fi) DESTINATION: Howson R Howson (B) ADDRESS: Spring House Corporate Center, P. 0. Box 457 (C) CITY: Spring House (D) STATE: Pennsylvama (E) COUNTRY: EU fl. (F) POSTAL CODE: 19477 (v) SHAPE TO BE READ BY COMPUTER: (fl) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: Compatible with IBM PC (C) OPTIFIfiTON SYSTEM: PC-DOS / MS-DOS ( D) APPLICATION PROGRAM: Patent Tn, Relay # 1.0, version # 1.25. (vi) DIFFERENCE OF THIS APPLICATION: (R) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vile) PREVIOUS APPLICATION DATA: (fl) APPLICATION NO .: 08 / 331,384 (B) DATE OF PRESENTATION: 28 -oct ubre- L994 (vin) EMPLOYEE INFORMATION / AGENT (A) NAME: Bak, rlary E. (ß) REGISTRATION NUMBER: 31,215 (C) REFERENCE / No. DE CRSO: GNVPN.007PCT (ix) TELECOMMUNICATIONS INFORMATION: (ft) TELEPHONE: 215-540-9200 (B) TELEFRX: 215-540-5818 (2) INFORMATION FOR SEQ ID NO: l: (i) FEATURES OF THE SEQUENCE: (A) LENGTH: 10,398 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: double (D) TOPOLOGY: unknown. Cu) TYPE OF MOLFCULR: oflDN (l) DESCRIPTION OF LR SEQUENCE: SEQ TD NO: 1: GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT 50 ATGATAATGA GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG 100 GGCGGGTGAC GTAGTAGTGT GGCGGAAGTG TGATGTTGCA AGTGTGGCGG 150 AACACATGTA AGCGACGGAT GTGGCAAAAG TGACGTTTTT GGTGTGCGCC 200 GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC GGATGTTGTA 250 GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT 300 GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA 350 ATATTTGTCT AGGGAGATCT GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC 400 CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA 450 GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC 500 TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA TTCGAGCTTG 550 CATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600 CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 650 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC 700 GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG 750 CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA 800 GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG_850_TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900 GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 950 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 1000 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 1050 GAGGTCTATA TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG 1100 ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA 1150 GCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAACTGAA AAACCAGAAA 1200 GTTAACTGGT AAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC 1250 GGTGGTGGTG CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT 1300 TCTAGGCCTG TACGGAAGTG TTACTTCTGC TCTAAAAGCT GCGGAATTGT 1350 ACCCGCGGCC GCAATTCCCG GGGATCGAAA GAGCCTGCTA AAGCAAAAAA 1400 GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA TTTTCGTTGC 1450 CGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500 ATCCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA 1550 CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA 1600 AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG 1650 AATGGCGCTT TGCCTGGTTT CCGGCACCAG AAGCGGTGCC GGAAAGCTGG 1700 CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC CCTCAAACTG 1750 GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800 TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC 1850 TCGCTCACAT TTAATGTTGA TGAAAGCTGG CTACAGGAAG GCCAGACGCG 1900 AATTATTTTT GATGGCGTTA ACTCGGCGTT TCATCTGTGG TGCAACGGGC 1950 GCTGGGTCGG TTACGGCCAG GACAGTCGTT TGCCGTCTGA ATTTGACCTG 2000 AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA TGGTGCTGCG 2050 TTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100 GCATTTTCCG TGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC 2150 GATTTCCATG TTGCCACTCG CTTTAATGAT GATTTCAGCC GCGCTGTACT 2200 GGAGGCTGAA GTTCAGATGT GCGGCGAGTT GCGTGACTAC CTACGGGTAA 2250 CAGTTTCTTT ATGGCAGGGT GAAACGCAGG TCGCCAGCGG CACCGCGCCT 2300 TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTÁTGCCG ATCGCGTCAC 2350 ACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400 ATCTCTATCG TGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT 2450 GAAGCAGAAG CCTGCGATGT CGGTTTCCGC GA JTGCGGA TTGAAAATGG 2500 TCTGCTGCTG CTGAACGGCA AGCCGTTGCT GATTCGAGGC GTTAACCGTC 2550 ACGAGCATCA TCCTCTGCAT GGTCAGGTCA TGGATGAGCA GACGATGGTG 2600 CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG TGCGCTGTTC 2650 GCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700 TGTATGTGGT GGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG 2750 AATCGTCTGA CCGATGATCC GCGCTGGCTA CCGGCGATGA GCGAACGCGT 2800 AACGCGAATG GTGCAGCGCG ATCGTAATCA CCCGAGTGTG ATCATCTGGT 2850 CGCTGGGGAA TGAATCAGGC CACGGCGCTA ATCACGACGC GCTGTATCGC 2900 TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG AAGGCGGCGG 2950 AGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000 ATGAAGACCA GCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG 3050 CTTTCGCTAC CTGGAGAGAC GCGCCCGCTG ATCCTTTGCG AATACGCCCA 3100 CGCGATGGGT AACAGTCTTG GCGGTTTCGC TAAATACTGG CAGGCGTTTC 3150 GTCAGTATCC CCGTTTACAG GGCGGCTTCG TCTGGGACTG GGTGGATCAG 3200 TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG CTTACGGCGG 3250 TGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300 TCTTTGCCGA CCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG 3350 CAGCAGTTTT TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG 3400 CGAATACCTG TTCCGTCATA GCGATAACGA GCTCCTGCAC TGGATGGTGG 3450 CGCTGGATGG TAAGCCGCTG GCAAGCGGTG AAGTGCCTCT GGATGTCGCT 3500 CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC AGCCGGAGAG 3550 CGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600 CATGGTCAGA AGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG 3650 GAAAACCTCA GTGTGACGCT CCCCGCCGCG TCCCACGCCA TCCCGCATCT 3700 GACCACCAGC GAAATGGATT TTTGCATCGA GCTGGGTAAT AAGCGTTGGC 3750 AATTTAACCG CCAGTCAGGC TTTCTTTCAC AGAT.TGGAT TGGCGATAAA 3800 AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG CACCGCTGGA 3850 TAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900 TCGAACGCTG GAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG 3950 CAGTGCACGG CAGATACACT TGCTGATGCG GTGCTGATTA CGACCGCTCA 4000 CGCGTGGCAG CATCAGGGGA AAACCTTATT TATCAGCCGG AAAACCTACC 4050 GGATTGATGG TAGTGGTCAA ATGGCGATTA CCGTTGATGT TGAAGTGGCG 4100 AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC AGCTGGCGCA 4150 GGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200 CCGACCGCCT TACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA 4250 GACATGTATA CCCCGTACGT CTTCCCGAGC GAAAACGGTC TGCGCTGCGG 4300 GACGCGCGAA TTGAATTATG GCCCACACCA GTGGCGCGGC GACTTCCAGT 4350 TCAACATCAG CCGCTACAGT CAACAGCAAC TGATGGAAAC CAGCCATCGC 4400 CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG ACGGTTTCCA 4450 TATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500 TACAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA 4550 TAATAATAAC CGGGCAGGCC ATGTCTGCCC GTATTTCGCG TAAGGAAATC 4600 CATTATGTAC TATTTAAAAA ACACAAACTT TTGGATGTTC GGTTTATTCT 4650 TTTTCTTTTA CTTTTTTATC ATGGGAGCCT ACTTCCCGTT TTTCCCGATT 4700 TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG GTATTATTTT 4750 TGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800 TTTCTGACAA ACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT 4850 GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA 4900 AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT ATTTGTAACC 4950 ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 5000 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT CCTCTAGAGT 5050 CGAGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT 5100 AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG 5150 CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA 5200 GTGAGCGAGC GAGCGCGCAG CAGATCTGGA AGGTGCTGAG GTACGATGAG 5250 ACCCGCACCA GGTGCAGACC CTGCGAGTGT GGCGGTAAAC ATATTAGGAA 5300 CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC GATCACTTGG 5350 TGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400 TGAGGTACTG AAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA 5450 AGGTGGGGGT CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG 5500 CCATGAGCAC CAACTCGTTT GATGGAAGCA TTGTGAGCTC ATATTTGACA 5550 ACGCGCATGC CCCCATGGGC CGGGGTGCGT CAGAATGTGA TGGGCTCCAG 5600 CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC TTGACCTACG 5650 AGACCGTGTC TGGAACGCCG TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700 GCCGCTGCAG CCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG 5750 CCCGCTTGCA AGCAGTGCAG CTTCCCGTTC ATCCGCCCGC GATGACAAGT 5800 TGACGGCTCT TTTGGCACAA TTGGATTCTT TGACCCGGGA ACTTAATGTC 5850 GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG CAGGTTTCTG CCCTGAAGGC 5900 TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA CCAGACTCTG 5950 TTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000 CGCGCGCGGT AGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG 6050 TATTTTTTCC AGGACGTGGT AAAGGTGACT CTGGATGTTC AGATACATGG 6100 GCATAAGCCC GTCTCTGGGG TGGAGGTAGC ACCACTGCAG AGCTTCATGC 6150 TGCGGGGTGG TGTTGTAGAT GATCCAGTCG TAGCAGGAGC GCTGGGCGTG 6200 GTGCCTAAAA ATGTCTTTCA CTAGCAAGCT GATTGCCAGG GGCAGGCCCT 6250 TGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTGGG 6300 GATATGAGAT GCATCTTGGA CTGTATTTTT AGGTTGGCTA TGTTCCCAGC 6350 CATATCCCTC CGGGGATTCA TGTTGTGCAG AACC? CCAGC ACAGTGTATC 6400 CGGTGCACTT GGGAAATTTG TCATGTAGCT TAGAAGGAAA TGCGTGGAAG 6450 AACTTGGAGA CGCCCTTGTG ACCTCCAAGA TTTTCCATGC ATTCGTCCAT 6500 AATGATGGCA ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG ATATTTCTGG 6550 GATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600 TTTACAAAGC GCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC 6650 CGGCCCAGGG GCGTAGTTAC CCTCACAGAT TTGCATTTCC CACGCTTTGA 6700 GTTCAGATGG GGGGATCATG TCTACCTGCG GGGCGATGAA GAAAACGGTT 6750 TCCGGGGTAG GGGAGATCAG CTGGGAAGAA AGCAGGTTCC TGAGCAGCTG 6800 CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT ACCGGGTGCA 6850 ACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900 ACTTCGTTAA GCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC 6950 CAGAAGGCGC TCGCCGCCCA GCGATAGCAG TTCTTGCAAG GAAGCAAAGT 7000 TTTTCAACGG TTTGAGACCG TCCGCCGTAG GCATGCTTTT GAGCGTTTGA 7050 CCAAGCAGTT CCAGGCGGTC CCACAGCTCG GTCACCTGCT CTACGGCATC 7100 TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC TTTCGCTGTA 7150 CGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200 GGCGCAGGGT CCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT 7250 CCGGGCTGCG CGCTGGCCAG GGTGCGCTTG AGGCTGGTCC TGCTGGTGCT 7300 GAAGCGCTGC CGGTCTTCGC CCTGCGCGTC GGCCAGGTAG CATTTGACCA 7350 TGGTGTCATA GTCCAGCCCC TCCGCGGCGT GGCCCTTGGC GCGCAGCTTG 7400 CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT TGAGGGCGTA 7450 GAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500 AGGCCCCGCA GACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT 7550 TCGGGGTCAA AAACCAGGTT TCCCCCATGC TTTTTGATGC GTTTCTTACC 7600 TCTGGTTTCC ATGAGCCGGT GTCCACGCTC GGTGACGAAA AGGCTGTCCG 7650 TGTCCCCGTA TACAGACTTG AGAGGCCTGT CCTuGACCGA TGCCCTTGAG 7700 AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC ATGACTATCG 7750 TCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800 CCGGCAGCGC TCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC 785s GACGATGATC GGCCTGTCGC TTGCGGTATT CGGAATCTTG CACGCCCTCG 7900 CTCAAGCCTT CGTCACTGGT CCCGCCACCA AACGTTTCGG CGAGAAGCAG 7950 GCCATTATCG CCGGCATGGC GGCCGACGCG CTGGGCTACG TCTTGCTGGC 8000 GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT CTTCTCGCTT 8050 CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100 GATGACGACC ATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG 8150 CCTAACTTCG ATCACTGGAC CGCTGATCGT CACGGCGATT TATGCCGCCT 8200 CGGCGAGCAC ATGGAACGGG TTGGCATGGA TTGTAGGCGC CGCCCTATAC 8250 CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT GCATGGAGCC GGGCCACCTC 8300 GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA CCACTCCAAG 8350 AATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400 CTTGGCAGAA CATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG 8450 CGCATCTCGG GCAGCGTTGG GTCCTGGCCA CGGGTGCGCA TGATCGTGCT 8500 CCTGTCGTTG AGGACCCGGC TAGGCTGGCG GGGTTGCCTT ACTGGTTAGC 8550 AGAATGAATC ACCGATACGC GAGCGAACGT GAAGCGACTG CTGCTGCAAA 8600 ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT TCCGTGTTTC 8650 GTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700 TCTGCATCGC AGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA 8750 TTAACGAAGC CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT 8800 AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 8850 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 8900 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 8950 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAA? »TGGTG GCCTAACTAC 9000 GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 9050 TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG 9100 CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 9150 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 9200 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 9250 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 9350 TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT 9400 GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 9450 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 9500 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 9550 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC 9650 AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG 9700 GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 9750 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 9800 AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA 9850 TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC 9950 ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG 10000 GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 10050 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 10100 10150 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG 10200 10250 CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 10300 10350 CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GTCTTCAA 10398 (2) INFORMATION PÍRfi SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (fi) LENGTH: 4910 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: double (D) TOPOLOGY: unknown . (Ii) MOLECULE TYPE: cDNA .. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 50 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 100 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG 150 CGGCATCAGA GCAGÁTTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 200 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT 250 CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 350 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGCCAA 400 GCTTGCATGC CTGCAGGTCG ACTCTAGAGG ATCCGAAAAA ACCTCCCACA 450 CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT 500 TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT 550, TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA 600 1 ACTCATCAAT GTATCTTATC ATGTCTGGAT CCCCGCGGCC GCCAAATCAT 650 TTATTGTTCA AAGATGCAGT CATCCAAATC CACATTGACC AGATCGCAGG 700 CAGTGCAAGC GTCTGGCACC TTTCCCATGA TATGATGAAT GTAGCACAGT 750 TTCTGATACG CCTTTTTGAC GACAGAAACG GGTTGAGATT CTGACACGGG 800 AAAGCACTCT AAACAGTCTT TCTGTCCGTG AGTG? AGCAG ATATTTGAAT 850 TCTGATTCAT TCTCTCGCAT TGTCTGCAGG GAAACAGCAT CAGATTCATG 900 CCCACGTGAC GAGAACATTT GTTTTGGTAC CTGTCTGCGT AGTTGATCGA 950 AGCTTCCGCG TCTGACGTCG ATGGCTGCGC AACTGACTCG CGCACCCGTT 1000 TGGGCTCACT TATATCTGCG TCACTGGGGG CGGGTCTTTT CTTGGCTCCA 1050 CCCTTTTTGA CGTAGAATTC ATGCTCCACC TCAACCACGT GATCCTTTGC 1100 CCACCGGAAA AAGTCTTTGA CTTCCTGCTT GGTGACCTTC CCAAAGTCAT 1150 GATCCAGACG GCGGGTGAGT TCAAATTTGA ACATCCGGTC TTGCAACGGC 1200CGAAGGTCGT TGAGTTCCCG TCAATCACGG CGCACATGTT 1250 GGTGTTGGAG GTGACGATCA CGGGAGTCGG GTCTATCTGG GCCGAGGACT 1300 TGCATTTCTG GTCCACGCGC ACCTTGCTTC CTCCGAGAAT GGCTTTGGCC 1350 GACTCCACGA CCTTGGCGGT CATCTTCCCC TCCTCCCACC AGATCACCAT 1400 CTTGTCGACA CAGTCGTTGA AGGGAAAGTT CTCATTGGTC CAGTTTACGC 1450 ACCCGTAGAA GGGCACAGTG TGGGCTATGG CCTCCGCGAT GTTGGTCTTC 1500 CCGGTAGTTG CAGGCCCAAA CAGCCAGATG GTGTTCCTCT TGCCGAACTT 1550 TTTCGTGGCC CATCCCAGAA AGACGGAAGC CGCATATTGG GGATCGTACC 1600 CGTTTAGTTC CAAAATTTTA TAAATCCGAT TGCTGGAAAT GTCCTCCACG 1650 GGCTGCTGGC CCACCAGGTA GTCGGGGGCG GTTTTAGTCA GGCTCATAAT 1700 CTTTCCCGCA TTGTCCAAGG CAGCCTTGAT TTGGGACCGC GAGTTGGAGG 1750 CCGCATTGAA GGAGATGTAT GAGGCCTGGT CCTCCTGGAT CCACTGCTTC 1800 TCCGAGGTAA TCCCCTTGTC CACGAGCCAC CCGACCAGCT CCATGTACCT 1850 GGCTGAAGTT TTTGATCTGA TCACCGGCGC ATCAGAATTG GGATTCTGAT 1900 TCTCTTTGTT CTGCTCCTGC GTCTGCGACA CGTGCGTCAG ATGCTGCGCC 1950 ACCAACCGTT TACGCTCCGT GAGATTCAAA CAGGCGCTTA AATACTGTTC 2000 CATATTAGTC CACGCCCACT GGAGCTCAGG CTGGGTTTTG GGGAGCAAGT 2050 AATTGGGGAT GTAGCACTCA TCCACCACCT TGTTCCCGCC TCCGGCGCCA 2100 TTTCTGGTCT TTGTGACCGC GAACCAGTTT GGCKAAGTCG GCTCGATCCC 2150 GCGGTAAATT CTCTGAATCA GTTTTTCGCG AATCTGACTC AGGAAACGTC 2200 CCAAAACCAT GGATTTCACC CCGGTGGTTT CCACGAGCAC GTGCATGTGG 2250 AAGTAGCTCT CTCCCTTCTC AAATTGCACA AAGAAAAGGG CCTCCGGGGC 2300 CTTACTCACA CGGCGCCATT CCGTCAGAAA GTCGCGCTGC AGCTTCTCGG 2350 CCACGGTCAG GGGTGCCTGC TCAATCAGAT TCAGATCCAT GTCAGAATCT 2400 GGCGGCAACT CCCATTCCTT CTCGGCCACC CAGTTCACAA AGCTGTCAGA 2450 AATGCCGGGC AGATGCCCGT CAAGGTCGCT GGGGACCTTA ATCACAATCT 2500 CGTAAAACCC CGGCATGGCG GCTGCGCGTT CAAACCTCCC GCTTCAAAAT 2550 GGAGACCCTG CGTGCTCACT CGGGCTTAAA TACCCAGCGT GACCACATGG 2600 TGTCGCAAAA TGTCGCAAAA CACTCACGTG ACCTCTAATA CAGGACTCTA 2650 GAGGATCCCC GGGTACCGAG CTCGAATTCG TAATCATGGT CATAGCTGTT 2700 TCCTGTGTGA AATTGTTATC CGCTCACAAT TCCACACAAC ATACGAGCCG 2750 GAAGCATAAA GTGTAAAGCC TGGGGTGCCT AATGAGTGAG CTAACTCACA 2800 TTAATTGCGT TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG 2850 CCAGCTGCAT TAATGAATCG GCCAACGCGC GGGGAGAGGC GGTTTGCGTA 2900 TTGGGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT 2950 CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC 3000 CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC 3050 AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 3100 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG 3150 GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT 3200 CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC 3250 GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCATAGCT CACGCTGTAG_3300_GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG 3350 AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 3400 GAGTCCAACC CGGTAAGACA CGACTTATCG CCAC'xGGCAG CAGCCACTGG 3450 TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA 3500 AGTGGTGGCC TAACTACGGC TACACTAGAA GGACAGTATT TGGTATCTGC 3550 GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA GCTCTTGATC 3600 CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTTGTT TGCAAGCAGC 3650 AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT 3700 ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT 3750 CATGAGATTA TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT 3800 GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT 3850 TACCAATGCT TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG 3900 TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT ACGATACGGG 3950 AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC 4000 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA 4050 GCGCAGAAGT GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT 4100 GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC 4150 GTTGTTGCCA TTGCTACAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT 4200 GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC 4250 CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC 4300 AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA 4350 TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG 4400 AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC 4450 TCTTGCCCGG CGTCAATACG GGATAATACC GCGCCACATA GCAGAACTTT 4500 AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA 4550 TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC 4600 TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC 4650 AGGAAGGCAA AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT 4700 GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT 4750 TATTGTCTCA TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA 4800 AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GACGTCTAAG 4850 AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG 4900 CCCTTTCGTC 4910

Claims (24)

NOVELTY OF Lñ INVENTION CLAIMS

1. - A hybrid, recombinant virus, with omission of reproduction, characterized in that it comprises: (a) adenovirus sequences comprising the adenovirus 5 'and 3' cis-ets, necessary for the reproduction and encapsidation of vinon; (b) adeno-associated virus sequences (VAfI) that com- ply 5 'and 3' inverted terminal (RTI) repeats of a VflR; said Vflfl sequences being flanked by the adenovirus sequences of (a), and (c) a selected gene, operably linked to the regulatory sequences which direct their expression in a target cell; said gene and said regulatory sequences are flanked by the Vflfl sequences of (b); wherein the hybrid virus is provided with sufficient adenovirus sequences to allow infection of a target cell and stable integration of the transgene into the target cell, in the presence of a functional portion of? n gene rep of Vflfl.

2. The hybrid virus according to claim 1, further characterized in that the adenovirus sequences comprise a functional omission in the El gene.

3. The hybrid virus according to claim 2, further characterized by the sequences of adenoviruses comprise a functional omission in the E3 gene.

4. The hybrid virus according to claim 1, further characterized in that the adenovirus sequences comprise functional omissions in one or more of the adenovirus genes, selected from the group consisting of: the E2a gene, the E4 gene, late genes Ll to L5; and intermediate genes IX and IV «.

5. The hybrid virus according to any of claims 1 to 4, further characterized in that the selected gene is a reporter gene.

6. Hybrid hybrid virus according to claim 5, further characterized in that the reporter gene is selected from the group consisting of the genes encoding beta-galactosidase, alkaline phosphatase and the green fluorescent protein.

7. The hybrid virus according to any of claims 1 to 4, further characterized in that the selected gene is a therapeutic gene.

8. The hybrid virus according to claim 7, further characterized in that the therapeutic gene is selected from the group consisting of a normal RTFC gene and a normal LDL gene.

9. The hybrid virus according to claim 1, further characterized in that it additionally comprises a gene rep of Vflfl.

10. A recornbinating hybrid vector, with omission of reproduction, characterized in that it comprises: (a) adenovirus sequences comprising the 5 'and 3' cis-elements of adenovirus, necessary for the reproduction and the virion encapsidation; (b) adeno-associated virus sequences (Vflfl) comprising the inverted terminal (RTT) 5 'and 3' repeats of an adeno-associated virus; said VAfl sequences being flanked by the adenovirus sequences of (a); (c) a selected gene, operatively linked to regulatory sequences directing their expression in a target cell; the gene and the regulatory sequences being flanked by the sequences of Vflfi do (b).

11. A recombinant transfection particle, characterized in that it comprises: (a) a recornbi ante hybrid virus, with omission of reproduction, comprising: (i) adenovirus sequences comprising the 5 'and 3 cis-energetics. of adenoviruses necessary for the reproduction and encapsulation of vipón; (n) adeno-associated virus (AAV) sequences comprising the 5 'and 3' reverse terminal repeats (RTI) of an adeno-associated virus; the AAV sequences being flanked by the adenovirus sequences of (i); and (m) a selected gene, operably linked to regulatory sequences that direct s? expression in a target cell; the gene and regulatory sequences being flanked by the VAfl sequences of (n); wherein the hybrid virus is provided with sufficient adenovirus sequences to allow the infection of a target cell and the stable integration of the transgene in the target cell, in the presence of a functional portion of a rep gene of AAV; (b) a polycation sequence, conjugated to the hybrid virus; and (c) a plasmid comprising a functional portion of a rep gene of Vflñ, operably linked to regulatory sequences capable of directing its expression; the plasmid being associated with the polycation sequence.

12. The transfection particle according to claim 11, further characterized in that the adenovirus sequences contain a functional omission in the gene.

13. The transfection particle of conformity with claim 11 or claim 12, characterized in addition, because the adenovirus sequences contain a functional omission in the E3 gene.

14. The transfection particle according to claim 11, further characterized in that the adenovirus sequences contain functional omissions in one or more of the adenovirus genes, selected from the group consisting of the E2a gene, the E4 gene, the late genes Ll to L5 and intermediary genes IX and IVa.

15. The transfection particle according to any of claims 11 to 14, further characterized in that the selected gene is a reporter gene.

16. The transfection particle according to claim 15, further characterized in that the reporter gene is selected from the group consisting of the genes encoding beta-galactosidase, alkaline phosphatase and green fluorescent protein.

17. The transfection particle according to any of claims 11 to 14, further characterized in that the selected gene is a therapeutic gene.

18. The particle according to claim 17, further characterized in that the therapeutic gene is selected from the group consisting of a normal RTFC gene and a normal LDL gene.

19. A composition for use in the supply and stable integration of a gene selected on the chromosome of a target cell, characterized in that said composition comprises: (a) a hybrid virus, recombinant, with omission of reproduction, that it comprises: (i) adenovirue sequences comprising the 5 'and 3' cis-elenentos of adenovirus, necessary for the reproduction and encapsidation of virions; (n) adeno-associated virus sequences (Vñfl) comprising the inverted terminal repeats (RTI) 5 'and 3' of an adeno-associated virus; said sequences of Vflfl being flanked by * the adenovirus sequences of (i); (iii) a selected gene, operatively linked to regulatory sequences that direct s? expression in a target cell; the gene and regulatory sequences being flanked by the Vññ sequences of (n); and (iv) a functional portion of a Vññ ba gene or control of regulatory sequences, capable of expressing the functional portion of the rep gene in the target cell; wherein the hybrid virus is provided with sufficient adenovirus sequences to allow infection of a target cell and stable integration of the transgene into the target cell, in the presence of a functional portion of the Vñfl gene rep; and (b) a pharmaceutically acceptable carrier. 20.- The composition in accordance with the r * e? vindication 19, further characterized in that the functional portion of the rep gene encodes the rep 78 and 52 proteins. 21. A composition for use in the delivery and the stable integration of a selected gene into the chromosome of a target cell, comprising a effective amount of a recombinant transfection particle in a pharmaceutically acceptable carrier; characterized in that said particle comprises: a hybrid recombinant virus, with omission of reproduction, which comprises: (i) adenovirus sequences comprising the 5 'and 3' cis-elements of adenovirus necessary for the reproduction and encapsidation of vipón; (n) adeno-associated virus (Vññ) sequences comprising the reverse terminal repeats (RTI) 5 'and 3' of an adeno-associated virus; the Vflñ sequences being flanked by the adenovirus sequences of (i); and (lii) a selected gene, operably linked to regulatory sequences that direct their expression in a target cell; the gene and regulatory sequences being flanked by the Vñfi sequences of (n); wherein the hybrid virus is provided with sufficient adenovirus sequences to allow infection of a target cell and stable integration of the transgene into the target cell, in the presence of a functional portion of the rep gene of VAfl; (b) a polycation sequence, conjugated to the hybrid virus; (c) a scam that comprises a functional portion of a VAR gene, operably linked to regulatory sequences capable of directing its expression; the embodiment being associated with the polycation sequence. 22. A mammalian cell capable of expressing a selected gene introduced therein by means of transduction of the virus of claim 1, the vector * of claim 10 and the transfection particle of claim 11. 23. - A method for producing high levels of a recornbinant adeno-associated virus, characterized in that it comprises the steps of: (a) culturing a cell cotransfected with the vector of claim 10 and an optional helper virus, in the presence of a plasmid containing a rep gene of VRfl, under the control of regulatory sequences, capable of expressing the rep gene; and (b) isolating a recombinant AAV from said culture. 24. - A method for producing high levels of a recombinant adeno-associated virus, characterized in that it comprises the steps of: (a) culturing a transfected cell with the transfection particle of claim 11; and (b) isolating a recombinant Vflfl from said culture,