CZ299418B6 - DNA construct, affecting expression of endogenous IFNA2 gene, isolated nucleic acid, homologous recombinant mammal cell, method of influencing expression of endogenous IFNA2 gene, and production process of IFNA2 - Google Patents

Abstract

The present invention relates to an isolated nucleic acid hybridizing under very strict conditions with a defined region disposed in a genome counter the direction of transcription relative to encoding sequence of IFNA2 gene, or which is identical by at least 80 percent with this sequence. The object of the proposed invention is further a DNA construct containing the indicated isolated DNA molecule as a targeting sequence for homologous recombination.

Description

DNA construct affecting expression of endogenous IFNA2 gene, isolated nucleic acid, homologous recombinant mammalian cell, method of affecting expression of endogenous IFNA2 gene, and method of producing IFNA2

Technical field

The present invention is concerned with genomic DNA.

BACKGROUND OF THE INVENTION

Proposed methods of treating diseases with the aid of therapeutic proteins include both the administration of proteins prepared in vitro and gene therapy. Generally, the preparation of a protein in vitro relates to the introduction of exogenous DNA encoding the corresponding protein into a suitable host cell in cell culture. Conversely, gene therapy methods involve administering to a patient cells, plasmids, or viruses containing a sequence encoding a corresponding therapeutic protein.

Some therapeutic proteins may also be produced by appropriately influencing the expression of their endogenous genes using targeted genetic engineering techniques. See, for example, U.S. Patent Nos. 5,641,670; 5,733,761 and 5,272,071; further, U.S. Patent Application Serial No. 08 / 406,030; and WO 91/06 666, WO 91/06 667 and WO 90/11 354, all of which are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention is based on the identification and sequencing of genomic 5 'DNA preceding the coding sequence of the human interferon-α 2 gene ("IFNA2"). This DNA can be used, for example, in a DNA construct which, upon integration into the genome of a cell via homologous recombination, alters (for example, increases) expression of the endogenous IFNA2 gene in mammalian cells. The term "endogenous gene for IFNA2" refers to the genomic (ie chromosomal) gene encoding IFNA2. The construct comprises a targeting sequence comprising or derived from a newly discovered non-coding 5 'and a sequence for regulating transcription. Preferably, the transcriptional regulatory gene sequence differs from the transcriptional regulatory sequence of the endogenous IFNA2 gene. The targeting sequence ensures the integration of the regulatory sequence into the region upstream of the endogenous IFNA2 coding sequence so that the regulatory sequence is operably linked to the endogenous coding sequence. The term "operably linked" refers to the fact that a regulatory sequence can direct the expression of an endogenous IFNA2 coding sequence. The construct may further comprise a selection marker, i.e., a gene region facilitating the selection of cells with a stably integrated construct and / or an additional coding sequence linked to a promoter.

In one embodiment, the DNA construct comprises: (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, (d) a splice donor site, (e) an intron, and (f) a splice acceptor site, wherein the targeting sequence provides integration and elements (b) - (f), wherein elements (b) - (f) are integrated into or upstream of the endogenous gene. The regulatory sequence then controls the production of transcripts that includes not only elements (c) to (f), but also endogenous IFNA2 coding sequence. Preferably, the intron and the splice acceptor site are located in the construct downstream of the splice donor site.

The targeting sequence is homologous to a preselected target sequence in the genome. Between these two homologous sequences, homologous recombination occurs. The targeting sequence comprises at least 20 (e.g., at least 30, 50, 100, or 1000) consecutive nucleotides of SEQ ID NO: 11, and may further comprise at least 20 (e.g., at least 30, 50, or

100) consecutive nucleotides of SEQ ID NO: 7, at least 20 (for example at least

Or 50) consecutive nucleotides of SEQ ID NO: 8, or at least 20 (e.g., at least 30, 50, 100 or 1000) consecutive nucleotides of SEQ ID NO: 12. contain at least 20 (for example at least 30, 50 or 100) consecutive nucleotides of SEQ ID NO: 15, at least 20 consecutive nucleotides of SEQ ID NO: 16, at least 20 (for example at least 30 or 50) consecutive nucleotides of the sequence SEQ ID NO: 17, or at least 20 (e.g., at least 30, 50, 100, or 1000) of consecutive nucleotides of SEQ ID NO: 18. SEQ ID NO: 7 corresponds to nucleotides 1 to 278 of SEQ ID NO: 11, SEQ ID NO: 8 corresponds to nucleotides 3492 to 3564 of SEQ ID NO: 11 and SEQ ID NO: 12 corresponds to nucleotides 279 to 3491 of SEQ ID NO: 11. The term "homologous" means that the targeting sequence is identical or sufficiently similar to its target site in the genome, so that homologous recombination can occur between the targeting sequence and the target site in a human cell. A small percentage of base pairs that do not pair with each other is acceptable if homologous recombination occurs at a sufficient frequency. To facilitate homologous recombination, it is preferred that the targeting sequence is at least 20 (e.g. at least

50, 100, 250, 400 or 1000) base pairs ("bp"). A targeting sequence may also include genomic sequences outside the region included in SEQ ID NO: 12 if it additionally comprises at least 20 nucleotides located within said region. For example, the complementary targeting sequence may be derived from a sequence lying between SEQ ID NO: 8 and endogenous transcription of the IFNA2 gene initiation sequence.

Due to the polymorphism of the IFNA2 gene locus, there may be minor changes in the nucleotide composition of any given target site in the genome in any existing mammalian species. The present invention also relates to targeting sequences which correspond to said polymorphic variants of SEQ ID NOs: 7, 8, 11, 12, 15, 16, 17 and 18 (especially variants corresponding to the polymorphism of the human IFNA2 gene).

By homologous recombination, the regulatory sequence of the construct integrates into the chromosome in a pre-selected region upstream of the coding sequence of the IFNA2 gene. The resulting novel transcriptional unit containing the regulatory sequence derived from the construct affects expression of the target30 IFNA2 gene. The IFNA2 protein thus produced may have a sequence identical to that of the IFNA2 protein that is encoded by the unaltered endogenous gene, or may contain additional, substituted or sparsely occurring amino acid residues, in addition to the wild-type IFNA2 protein, due to changes introduced by homologous recombination.

Effects on gene expression include activating (or causing expression) of a gene that is normally silent in the parent cell (i.e., not expressed), increasing or decreasing the level of gene expression, and changing the mode of gene regulation so that it is different from that of the original cell. The term "parent cell" refers to a cell prior to homologous recombination.

The present invention further provides a method of using the proposed DNA construct to effect expression of an endogenous IFNA2 gene in mammalian cells. The method comprises the steps of: i) introducing the DNA construct into mammalian cells, ii) maintaining the cells under conditions that allow homologous recombination between the construct and a target site in the genome (i.e., a site in the genome that is homologous to the targeting DNA sequence of the construct), and thereby also allow for the formation of homologously recombinant cells; and iii) maintaining homologously recombinant cells under conditions that allow expression of the IFNA2 coding sequence that is under the control of a regulatory sequence derived from the DNA construct. At least a portion of the target site in the genome is located 5 'to the coding sequence of the endogenous IFNA2 gene. That is, the target site in the genome may contain a coding sequence as well as a 5 'non-coding sequence.

The invention also encompasses transfected or infected cells in which homologous recombination has taken place between the construct and genomic DNA at an upstream site from the endogenous ATG initiation codon, in one or both alleles of the endogenous IFNA2 gene. Such transfected or infected cells, also called homologously recombinant cells, have an altered way of expressing the IFNA2 gene. Such cells are particularly suitable for in vitro

The production of IFNA2 and for the transfer of IFNA2 by the gene therapy method. Methods for preparing and using such cells are also within the scope of the present invention. The cells are of vertebrate origin, most often cells of mammalian origin (for example human, and may be derived from primate cells, cow cells, pig cells, equine cells, goat cells, sheep cells, feline dogs, rabbits, mice, guinea pigs, hamsters or rats).

The invention further relates to a method for producing mammalian IFNA2 protein in vitro or in vivo by the method of introducing the above construct into a host cell and inserting the construct into the genome of said cell via homologous recombination. Further, the homologously recombinant cell is maintained under conditions that allow transcription, translation and possibly IFNA2 protein secretion.

The invention further provides an isolated nucleic acid comprising a sequence of at least 20 (for example at least 30, 50, 100, 200 or 1000) of consecutive nucleotides of SEQ ID NO: 11 or the corresponding complementary sequence or sequence identical to SEQ ID NO: 11 except polymorphic. variations or other minor variations (e.g., less than 5% of the sequence) that do not prevent homologous recombination with the target sequence. For example, the isolated DNA may comprise at least 20 (e.g., at least 30, 50 or 100) consecutive nucleotides of SEQ ID NO: 7 or the corresponding complementary sequence, at least 20 (e.g., at least 30 or 50) consecutive nucleotides of SEQ ID NO: 8 or the corresponding a complementary sequence of at least 20 (e.g., at least 30, 50, 100 or 1000) of consecutive nucleotides of SEQ ID NO: 12 or a corresponding complementary sequence of at least 20 (e.g. of at least 30, 50 or 100) of consecutive nucleotides of SEQ ID NO: 15 or a corresponding complementary sequence of at least 20 contiguous nucleotides of SEQ ID NO: 16 or a corresponding complementary sequence of at least 20 (e.g. at least 30 or 50) of contiguous nucleotides of SEQ ID NO: 16;

NO: 17 or the corresponding complementary sequence or at least 20 (e.g., at least 30, 50, 100 or 1000) of consecutive nucleotides of SEQ ID NO: 18 or the corresponding complementary sequence.

In one embodiment of the present invention, the nucleic acid of the invention comprises a block

Thus, the isolated DNA may comprise, for example, nucleotides 1 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 700, 800, 801 to 900, 901 to 1000, 1001 to 1100, 1101 to 1200, 1201 to 1300, 1301 to 1400, 1401 to 1500, 1501 to 1600, 1601 to 1700, 1701 to 1800, 1801 to 1900, 1901 to 2000, 2001 to 2100, 2101 to 2200, 2201 to 2300, 2301 to 2400, 2401 to 2500, 2501 to 2600, 2601 to

2700 to 2800, 2801 to 2900, 2901 to 3000, 3001 to 3100, 3101 to 3200, 3201 to 3300,

3301 to 3400, 3401 to 3500 or 3465 to 3564 of SEQ ID NO: 11 or the corresponding complementary sequence. Said blocks of SEQ ID NO: 11 or corresponding complementary sequences are also suitable as targeting sequences for the constructs of the invention.

As isolated DNA, the sequence derived from SEQ ID NO: 11 is not linked to a sequence encoding intact IFNA2, or at least is not linked in the same configuration (i.e., separated by the same non-coding sequence) as in the wild-type genome. Thus, the term "isolated DNA" as used herein does not refer to a chromosome or large portions of genomic DNA (which could be incorporated into a cosmid or an artificial yeast chromosome) comprising not only a partial or all of SEQ ID NO: 11 but also an intact sequence encoding IFNA2. and the entire sequence lying between the IFNA2 coding sequence and the sequence corresponding to SEQ ID NO: 11 as it occurs in the intact cell genome. The term includes (but is not limited to) DNA which i) is incorporated into plasmids or virus, or ii) exists as a separate molecule independent of other sequences, for example a polymerase chain reaction (PCR) fragment or frag50 ment prepared by restriction endonuclease digestion. Preferably, the isolated DNA does not contain a sequence encoding an intact IFNA2 precursor (i.e., complete 1FNA2 together with an endogenous signaling secretory peptide).

The invention further provides an isolated DNA comprising a strand comprising at least 100 (e.g. at least 200, 400 or 1000) nucleotides long sequence,

Or very stringent conditions with SEQ ID NOs: 7, 8, 11, 12, 15, 16, 17 and / or 18 or with sequences complementary to SEQ ID NOs: 7, 8, 11, 12, 15 , 16, 17 and / or 18. The sequence is not linked to the IFNA2 coding sequence, or at least is not linked in the same configuration as in the wild-type genome. Slightly stringent conditions indicate hybridization at 50 ° C in Church Buffer (7% SDS, 0.5% NaHPO 4, 1 M EDTA, 1% bovine serum albumin) and washing at 50 ° C in 2x SSC. High stringency conditions are defined as: hybridization at 42 ° C in the presence of 50% formamide; a first wash at 65 ° C in 2x SSC with the addition of 1% SDS and a second wash at 65 ° C in 0.1x SSC.

Further provided is an isolated DNA comprising a strand having a sequence of (1) at least 50 (e.g. at least 70 or 100) nucleotides in length and (2) at least 80% (e.g. at least 85%, 90%, 95% or 98 %) identical to the fragment or all of SEQ ID NO: 11 or the corresponding complementary sequence. The fragment may comprise, for example, part or all of SEQ ID NO: 7, 8, 12, 15, 16, 17, 18 or 19. The sequence is not linked to an intact IFNA2 coding sequence, or at least is not linked in the same configuration as in the wild-type genome. .

Talking about a certain percent identity or conservation of a particular polypeptide or nucleic acid molecule relative to a reference polypeptide or nucleic acid, this percent identity or conservation is determined using an algorithm defined by Myers and Milller, CABIOS (1989), which is part of the ALIGN program 2.0), or equivalent, using the following parameters (if required): “gap length penalty” (ie, gap penalty) 12 and “gap penalty” (ie, gap penalty) 4. For all other default values were used. Program

ALIGN is easily accessible. See. for example, the Internet address: http://www2.igh.cnrs.fr/bin/align-quess.cgi.

The invention further relates to a method of delivering IFNA2 to the body of an animal (e.g., a mammal such as a human, other primate, cow, pig, horse, goat, sheep, cat, dog, rabbit, mouse, guinea pig, hamster or rat). that the pre-prepared cells whose endogenous 1FNA2 gene has been activated as described above are implanted in the body of said animal. The implanted cells then produce and secrete IFNA2 in the body of the animal. The present invention also provides a method of producing IFNA2 by pre-preparing cells whose endogenous IFNA2 gene has been activated as described above, cultured in vitro under conditions that allow the cells to express and secrete IFNA2.

The invention further includes isolated DNA that is at least 80% (for example at least 85%, 90% or 95%) identical or hybridizes under high or slightly stringent conditions to a portion (for example at least 20, 50, 100, 400 or 1000 base pairs long) The HindIII-BamHI insert of the plasmid pA2HB plasmid (described below). The 3 'end of said portion of this insert is located at least 511 bp upstream of the translation initiation codon of the ATG coding sequence of IFNA2 that is part of said insert in the plasmids.

The isolated DNA of the invention can be used, for example, as a source of upstream PCR primers. These primers can be used (in combination with suitable downstream primers) to obtain the regulatory region sequence and / or the complete coding sequence of the endogenous IFNA2 gene. Further, this DNA can be used as a hybridization probe to detect the presence of chromosome 9 in a human chromosome sample, or as described below, to affect expression of endogenous IFNA2 gene in vertebrate cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Examples of methods and materials used are given below, but methods and materials similar or equivalent to those described may also be used in the practice of the invention or its testing. All publications, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety. In case of conflict, the specification of the present invention, including definitions, is critical. The materials, methods and examples are illustrative only and in no way limit the scope of the invention.

Other features and advantages of the present invention will appear from the following detailed description of the invention and from the claims.

The present invention is based on the discovery of the nucleotide composition of the sequence upstream of the coding sequence of the human IFNA2 gene.

Interferon-α forms a complex gene family consisting of a cluster of 14 genes on the short arm of the 9th chromosome. None of these genes, including the IFNA2 gene, contain introns. Interferon-α is produced by macrophages, B and T cells and a variety of other cells. Interferon-α has significant antiviral effects and has been shown to be effective in the treatment of infections caused by papilloma virus, hepatitis B and C viruses and vaccinia, herpes simplex virus, herpes zoster varicellosus virus and rhinovirus.

The human 1FNA2 gene encodes a 188 amino acid long precursor protein (SEQ ID NO: 2) containing a 23 amino acid long signal peptide. The genomic map of the human IFNA2 gene is shown in Figure 1. The map is constructed based on the published 1733 bp long sequence (HUMIFNAA, GenBank, accession numbers J00207 and V00544; SEQ ID NO: 1) starting at position -510 relative to the translational start (unless otherwise stated, all positions listed herein are numbered relative to the translation start site) and terminate at position +1223. The CAP site is located at position -67.

Specific sequences located 5 'to the IFNA2 gene and their use to influence the expression of endogenous IFNA2 gene.

Genomic DNA containing the upstream sequence with respect to the IFNA2 gene was obtained as follows: The human leukocyte genomic library in lambda EMBL3 (Clontech catalog # HL1006d) was tested using a 332 bp long probe prepared by PCR. This probe corresponds to the genomic region of -263 to +69 and was amplified from human genomic DNA using the oligonucleotide primers IFN7 and IFN6. Both primers were designed based on the available IFNA2 genomic DNA sequence (Fig. 1). The 5 'end of primer IFN7 corresponds to position -263 and the sequence of this primer is 5-AGTTTCTAAAAAGGCTCTGGGGTA-3' (SEQ ID NO: 3). The 5 'end of primer IFN6 corresponds to position +69 and the sequence of this primer is 5'-GCCCACAGAGCAGCTTGAC-3' (SEQ ID NO: 4).

Approximately one million recombinant phages were tested using a radiolabeled 322 bp probe. 60 positive plaques were isolated from the primary test plates. The lambda phage DNA was isolated from thirty of these positive plaques. DNA was isolated by a template in a PCR reaction with oligonucleotide primers IFN1 and IFN2. Both primers IFN1 and IFN21 are derived from the 3 'untranslated region of the IFNA2 gene; their sequences can be found at "http://www.ncbi.nlm.nih.gov/dbSTS" under the identification code "NCBIID: 42433". The 5 'end of primer IFN1 corresponds to position +639 and the sequence of the primer is 5-AAAGACTCATGTTTCTGCTATGAC-3' (SEQ ID NO: 5). The 5 'end of primer IFN2 corresponds to position +853 and the sequence of the primer is 5-GGTGCACATGACATAATATGAACA-3' (SEQ ID NO: 6). Of the thirty phage clones tested, two were the expected PCR product of 215 bp. One of these two phage plaques was further purified in the other two rounds of hybridization experiments. Thus, a 4-4-1 phage clone was obtained. An 8.3 kb HindIII-BamHI phage fragment 4-4-1 was subcloned into plasmids pBluescript II SK + (Stratagene, La Jolla, CA) to give pA2HB. The pA2HB construct carries an approximately 4.3 kb portion of the untranscribed sequence upstream, a protein coding region (1.1 kb), and an approximately 2.8 kb portion of the downstream sequence of the IFNA2 gene. A restriction map of the 8.3 kb HindIII-BamHI fragment is shown in Figure 2.

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Plasmid pA2HB was sequenced by the Sanger method. 278 bp long sequence whose 5 'end is also the 5' end of the Hind III restriction site is shown below (see also FIG. 3) AAGCTTTTATAGTTGTAAATTTTCCACTTAGTACTGCTTTTGTAATGTTGTCTT TTTATTTTCATTTATCTCAAGATGTTTTCTAATTTCTCTTGACTTCCTTCTTAA ATTCTTACCTCATGTAGACATACATTTTTGGCCCTATGCATTGGGATGCAAAA CCAGACTAATTTACTTTGTACAAAAAGAAAAATGAGAAAGAAATATATTTGG TCTTGTGAGCACTATATGGAAATACTTTATATTCCATTTGTTTCATCATATTCA TATATCCCTTT (SEQ ID NO: 7)

The HindIII site is located at position 4,073. The previously unpublished sequence between positions 583 and -511 was also determined and is shown below, and is further highlighted by the underline in Figure 4.

CATTGGATACTCCATCACCTGCTGTGATATTATGAATGTCTGCCTATATAAAT ATTCACTATTCCATAACACA (SEQ ID NO: 8) 10 The sequence (SEQ ID NO: 12) located between regions of SEQ ID NO: 7 and SEQ ID NO: 8 was also determined.

The genomic sequence found at positions -4,074 to -511 (SEQ ID NO: 11) is that which is not underlined in Figure 6. SEQ ID NO: 7 and SEQ ID NO: 8 correspond to nucleotides 1-278 and 3492-3564 of SEQ ID NO: 11, respectively.

In order to influence the expression of the endogenous IFNA2 gene, the DNA fragment containing nucleotides 279-3311 of SEQ ID NO: 12 was cloned into a plasmid to produce the targeting construct pGA402. The nucleotide sequence 279-3311 of SEQ ID NO: 11 was named SEQ ID NO: 13. The fragment was inserted into the vector at a site upstream of the CM V promoter and neomycin resistance gene (see scheme in Figure 5). The second targeting sequence (see Figure 5), containing the DNA fragment of the 1FNA2 -68 to 69 gene (see Figure 1), was cloned into a plasmid downstream of the CMV promoter and neomycin resistance encoding gene.

The portion of the nucleotide sequence of the IFNA2 gene delimited by nucleotides -68 to 69 was named SEQ ID NO: 14. Plasmid pGA402 was introduced into human fibroblast cells displaying little or no expression of the IFNA2 gene to allow homologous recombination with the endogenous IFNA2 gene. Cells that were resistant to G418 after transformation with a plasmid were further tested to identify cells overexpressing the IFNA2 gene. This overexpression was expected when homologous recombination occurred between pGA402 and genomic DNA in close proximity to the endogenous IFNA2 gene.

General methodology:

Effect on endogenous expression of IFNA2 gene

Using the above-described upstream sequences from the IFNA2 gene, it is possible to influence the expression of an endogenous human IFNA2 gene as described in U.S. Patent No. 5,641,670. One of the strategies is shown in Figure 5. In this case, the targeting construct is designed to comprising a first targeting sequence homologous to a first target site located upstream of the gene, an amplifiable trait gene, a selectable marker gene, a regulatory region, a CAP site, an exon, a splice donor site, an intron, a splice acceptor site, and a second targeting sequence homologous to a second target a site located downstream of the first target site and terminating either in or upstream of the IFNA2 coding sequence. In accordance with this strategy, the 5 'end of the second target site is preferably positioned upstream of the transcription direction less than 107 bp away from the normal IFNA2 initiation initiation site. Thus, unwanted ATG start codons within the transcribed sequence can be avoided. The locus transcript prepared by the described homologous recombination comprises an exon, splice donor site, intron, splice acceptor site (all originating from the construct), any sequence between these elements, and a sequence from the splice acceptor site through the entire endogenous coding sequence to transcriptional termination site of the IFNA2 gene. Splicing of such transcripts results in an mRNA that is translated to produce a precursor of human IFNA2 containing, depending on the characteristics of the construct-encoded exon, either a normal secretion signal sequence or a genetically engineered secretion signal sequence. The size of the exogenous β intron and hence the position of the exogenous regulatory region relative to the coding region of the gene is variable and its changes can be used to optimize the function of the regulatory region.

For any activation strategy, it is not necessary for both target sites to be closely adjacent or closely adjacent. If these sites are not closely adjacent, a portion of the upstream region of the normal IFNA2 gene and / or a portion of the coding region is removed by homologous recombination.

By the method of homologous recombination, mutations can be introduced into the chromosome DNA to facilitate the expression of endogenous IFNA2. For example, it may be desirable to abolish an inappropriate and unwanted ATG initiation codon located upstream of the correct ATG initiation codon and between the exogenous regulatory region and the endogenous IFNA2 coding region at the homologously recombinant locus. For this purpose, a targeting construct having a targeting sequence homologous to a site in the genome that includes an undesired ATG initiation codon may be utilized. This targeting sequence comprises nucleotides that carry the desired mutation, for example an ATT site of the ATG. The targeting construct may comprise one or more selection traits to facilitate selection of homologously recombinant cells. The exogenous regulatory region can then be introduced into homologously recombinant cells upstream of the altered sites using the expression control method of the invention.

Alternatively, the exogenous regulatory region and the desired mutated sequence (s) may be inserted into the genome in a single step. The DNA construct used for this type of application contains both an exogenous regulatory region and a targeting sequence that contains the desired mutation (s). Another way is to co-transfect or co-infect target cells with two different constructs, where one construct contains a regulatory region and the other construct carries the desired mutation.

If desired, a mammalian splice acceptor site can also be introduced into the genome in a similar manner, for example, into the region between the undesired ATG initiation codon and the correct ATG initiation codon. For this purpose, the DNA construct comprises a targeting sequence homologous to the genomic region upstream of the correct IFNA2 initiation codon and a sequence corresponding to the desired splice acceptor site that is directly part of or adjacent to the homologous region. Cells containing the correct recombined IFNA2 locus are then transfected or infected with a second construct comprising an exogenous regulatory region and an exon with an unpaired splice donor site at its 3 'end together with a targeting sequence (or sequences) that direct the second construct to the genomic region upstream relative to the previously introduced splice acceptor site. The primary transcript produced under the control of the exogenous regulatory region includes an exogenous exon, an exogenous splice donor site, an exogenous splice acceptor site, any sequence between these elements, and a sequence between the exogenous splice acceptor site and the transcription termination site of the endogenous IFNA2 gene. By splicing, the splice donor site in the transcripts and the splice acceptor site approach and the intron RNA lying between them, which may contain unwanted AUG initiation codons, will be excised. All problems associated with the existence of undesirable AUG translation initiation codons in transcripts in the region between the transcriptional start and the IFNA2 coding sequence are thus eliminated. Of course, the regulatory region, exon, splice donor site, and splice acceptor site can be introduced into the genome in a single step. The DNA construct used for this application comprises a regulatory region, exon, splice donor site, intron, splice acceptor site directing a sequence homologous to the genomic region between the correct

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IFNA2 is an initiation codon and an undesired ATG codon, and optionally also one or more selection markers. Alternatively, two independent targeting constructs may be suitable, one containing the regulatory region, the exon and the splice donor site, and the other comprising the splice acceptor site. Both constructs can be introduced into target cells in a single step.

DNA construct.

The DNA construct of the invention comprises at least a targeting sequence and a regulatory sequence. In addition, it may optionally contain an exon; or an exon and a splice donor site; or an exon, splice donor site, intron, and splice acceptor site. If the construct is an exon, it is located at the 3 'end of the regulatory region, and if the construct is a splice donor site, it is located at the 3' end of the exon. The intron and the splice acceptor site, if present in the construct, are located at the 3 'end of the splice donor site. Further, the construct may contain multiple exons and introns (with corresponding splice donor and acceptor sites). The DNA in the construct is referred to as exogenous, since this DNA is not an original part of the host cell genome. The exogenous DNA may comprise sequences identical or different to the endogenous genomic DNA present in the cell prior to transfection or infection with the viral vector. As used herein, the term "transfection" refers to the introduction of plasmids into a cell by a non-viral (ie, chemical or physical) method such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, microprojectiles etc. The term "infection" refers to the introduction of a viral vector into a cell by a viral infection method. The various elements that are part of the DNA construct of the invention are described in more detail below.

The DNA construct may further comprise cis- and / or β-acting viral sequences (e.g., envelope signals) allowing delivery of the construct to the cell nucleus by a viral vector infection. If necessary, the DNA construct can be detached from some parts of the viral life cycle, such as integrating a retrovirus into the genome by integrase or maintaining an episome in the cell. This excision can be achieved by corresponding deletions or mutations of the viral 30 sequences, for example, deletion of the integrase coding region in retroviral vectors. Further details regarding the construction and use of viral vectors are given, for example, in Robbins et al., Pharmacol. Ther. 80: 35-47, 1998 and Gunzburg et al., Mol. Copper. Today 1: 410-417, 1995, which are incorporated herein by reference.

Routing sequence.

The targeting sequences allow homologous recombination of the desired sequence into a selected region of the host genome. The targeting sequences are homologous (ie capable of homologous recombination) with their corresponding target sites in the host genome.

The circular DNA construct may comprise a single targeting sequence, or two or more separate targeting sequences. The linear DNA construct may comprise two or more separate targeting sequences. The target site with which a given targeting sequence is homologous can be located within the coding region of the IFNA2 gene, upstream of and closely adjacent to the coding region or upstream but remote from the coding region.

The first of the two targeting sequences in the construct (or the entire targeting sequence if the construct contains only one targeting sequence) is derived from the newly described genomic regions upstream of the IFNA2 coding region. This routing sequence comprises a portion (e.g.

20 or more consecutive nucleotides) of SEQ ID NO: 11, for example a portion of SEQ ID NO: 7, 8 or 12. The second of the two targeting sequences in the construct may be directed against the genomic region upstream of the coding sequence, or may be directed against part or all of the coding sequence. For example, the second targeting sequence may comprise at its 3 'end an "exogenous" coding region identical to the first few codons of the IFNA2 coding sequence. Homologous recombination recombines the exogenous coding region with an endogenous portion

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IFNA2 coding sequence. If desired, the exogenous coding region may encode a heterologous amino acid sequence as long as the exogenous coding region remains sufficiently homologous to the endogenous coding sequence it replaces to allow homologous recombination between them.

The targeting sequence may additionally include sequences derived from the previously described region of the IFNA2 gene, including those described herein, as well as from a region located even further upstream, whose structure has not yet been described, but which can be characterized by those skilled in the art.

Genomic fragments suitable for use in the function of targeting sequences may be identified by their ability to hybridize to a probe comprising all or part of the sequence of SEQ ID NO: 11. Such a probe may be prepared by PCR using primers derived from SEQ ID NO: 11.

Regulatory sequence.

The regulatory sequence of a DNA construct may comprise one or more promoters (e.g., a constitutive, tissue-specific or inducible promoter), enhancers, scaffold-attachment regions or matrix-binding sites, negative-regulatory elements, binding sites for transcription factors or a combination of these elements.

The regulatory sequence may be derived from a eukaryotic (e.g., mammalian) or viral genome. Suitable regulatory sequences include, but are not limited to, sequences that regulate SV40 expression of early or late genes, cytomegalovirus genes, and major late adenovirus genes. This group also includes regulatory regions derived from genes encoding mouse metallothionein-1, elongation factor-ία, collagen (e.g., collagen Ia1, collagen Ia2 and collagen IV), actin (e.g., γ-actin), immunoglobulin, HMG-CoA reductase, glyceraldehyde phosphate dehydrogenase, 3-phosphoglycerate kinase, collagenase, stromelysin, fibronectin, vimentin, plasminogen I activator inhibitor, thymosin B4, tissue metalloproteinase inhibitors, ribosomal proteins, major histocompatibility complex molecules, and human leukocyte antigens.

The regulatory sequences preferably comprise a binding site for transcription factors such as

TATA box, CCAAT box, API, Spi or NF-κΒ binding site.

Indicator genes.

If desired, the construct may comprise a sequence encoding a polypeptide of interest, operably linked to its own promoter. An example may be a selectable marker gene that is used to facilitate identification of a linker targeting sequence with its target sequence. The amplifiable trait gene may also be part of the construct and is used to facilitate selection of cells with co-amplified adjacent DNA sequences. Cells containing amplified copies of the amplifiable trait are recognized by growth in the presence of a substance that differentiates cells expressing the amplifiable gene. The activated endogenous IFNA2 gene is amplified in tandem along with an amplifiable selection marker. Cells containing multiple copies of an activated endogenous gene can produce very high concentrations of IFNA2 and are therefore suitable for in vitro protein production or gene therapy.

The genes for the selectable and amplifiable trait do not necessarily lie in close proximity. The selectable marker gene may be identical to the amplifiable marker gene. One or both of the identifying genes may be part of an intron of the DNA construct. Suitable identifying genes of both types are described in U.S. Patent No. 5,641,670.

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Donor splice site and acceptor splice site.

The DNA construct may further comprise an exon, a splice donor site at the 3 'end of the exon, an intron, and a splice acceptor site.

The splice donor site is a sequence that directs splicing of one exon of the RNA transcript to the acceptor site of another exon of the transcript. The result of splicing is the removal of the intron between the two sites. Typically, the first exon lies 5 'with respect to the second exon and the splice donor site located at the 3' end of the first exon is paired with the acceptor splice site adjacent to the second exon at its 5 'end. The splice donor sites have a characteristic conserved sequence (A / C) of AGGURAG (where R represents a purine nucleotide), wherein

GUs at positions 4 and 5 are necessary (Jackson, Nucleic Acids Research 19: 3715-3798, 1991). The first three nucleotides of the conserved splice donor site are also the last three nucleotides of the exon: i.e., they are not cut. The splice donor sites are functionally defined based on their ability to carry out a corresponding reaction within the mRNA splice pathway.

The splice acceptor site in the construct of the invention provides, together with the splice donor site, splicing of the intron between two exons. The splice acceptor site has the characteristic sequence (Y) ₁₀ NYAG (SEQ ID NO: 19) wherein Y represents any pyrimidine and N represents any nucleotide (Jackson, Nucleic Acids Research 19: 3715-3798, 1991).

CAP instead.

The DNA construct may optionally also contain a CAP site. The CAP site is a specific transcriptional start that is associated with and utilized by the regulatory region. The CAP site is located in the construct with respect to the regulatory sequence such that after homologous recombination, the regulatory sequence directs the synthesis of a transcript that begins at the CAP site. Alternatively, the CAP site may not be part of the construct, and then the transcriptional machinery implicitly locates the corresponding site in the target gene, and this is then used as the CAP site.

Other DNA elements.

The construct may further comprise sequences that affect the structure or stability of the RNA or protein prepared by homologous recombination.

Optionally, the DNA construct may comprise a bacterial origin of replication and some bacterial selection marker, for example an antibiotic resistance gene. This allows the plasmids to multiply in bacteria or other suitable large-scale cloning / host system.

All of the above-described elements of the DNA construct are operably linked or functionally positioned within the construct with respect to each other. That is, due to homologous recombination between the construct and the target site in the genome, the regulatory sequence can direct the production of a primary RNA transcript that begins at the CAP site (optionally, the CAP site may be part of the construct) and includes a sequence lying between the CAP site and endogenous STOP-codon. IFNA2 gene.

Depending on the location of the CAP site, this sequence may include a portion of the endogenous regulatory region of the IFNA2 gene, as well as sequences that are adjacent to this region and are not normally transcribed. If the construct contains an exon and a donor and acceptor splice site, then the primary transcript also includes an exon, two splice sites, and an intron lying between the two sites.

The order of the individual elements in the DNA construct may be variable. If the construct is a circular plasmid or a viral vector, the relative order of the individual elements in the resulting structure is, for example: the targeting sequence, the plasmid DNA (containing the sequences used for

Selection and / or replication of targeting plasmids in a microbial or other suitable host), a selection facilitating sequence (a so-called selection marker), a regulatory sequence, an exon, a splice donor site, an intron, and an acceptor splice site.

For example, if the construct is linear, the sequence of the individual elements is as follows: first targeting sequence, selection marker, regulatory sequence, exon, splice donor site, intron, splice acceptor site, and second targeting sequence, or, alternatively, first targeting sequence, regulatory sequence, exon, splice donor site, intron, splice acceptor site, selection marker, optionally internal ribosome-binding site, and finally a second targeting sequence.

Alternatively, the sequence may be as follows: first targeting sequence, first selection marker, regulatory sequence, exon, splice donor site, intron, splice acceptor site, second targeting sequence, and second selection feature; or a first targeting sequence, a regulatory sequence, an exon, a splice donor site, an intron, a splice acceptor site, a first selection marker, a second targeting strand cleavage, and a second selection marker.

Recombination between targeting sequences adjacent to the first selection marker and homologous sequences in the host genome results in a directed integration of the first selection marker, while the second selection marker is not integrated. Desirable transfected or infected cells are those that are stably transfected or infected with a first but not a second selection marker. Such cells may be selected by growth in a medium containing a substance that favors cells expressing the first selection marker and a second substance that selects for cells expressing the second selection marker. Transfected or infected cells with an improperly integrated targeting construct (i.e., cells that have been integrating the construct by a mechanism other than homologous recombination) will also express a second selection trait and thus be killed in the selection medium.

In some instances, a construct for positive selection is included in the construct, that is, one that allows the selection of cells containing amplified copies of the trait. In such a case, the order of the elements in the construct is, for example, as follows: first targeting sequence, amplifiable positive selection marker, (optionally) second selection marker, regulatory sequence, exon, splice donor site, intron, splice acceptor site and second targeting sequence.

Individual elements of the DNA construct may be obtained either from natural sources (eg, genomic DNA), or may be obtained by genetic engineering methods, or may be synthetically prepared. The regulatory region, CAP site, splice donor site, intron, and splice acceptor site can be isolated as a complete unit, for example, from the human elongation factor-ία gene (Genbank, sequence HUMEF1A) or from the region of the very early cytomegalovirus genes (Genbank, HEHCMVP1) . However, these components can also be isolated from individual genes.

Transfection or infection and homologous recombination.

The DNA construct of the invention can be introduced into a cell, for example a primary, secondary or immortalized cell line, as a single DNA construct or in the form of separate DNA sequences that are incorporated into the chromosomal or nuclear DNA of the transfected or infected cell. The term "transfected cell" refers to a cell into (or whose ancestor) a DNA or RNA molecule has been introduced in a manner other than using a viral vector. The term "infected cell" refers to a cell into which (or to whose ancestor) a molecule has been introduced

DNA or RNA using a viral vector. Viruses suitable for use in the function of vectors include adenoviruses, adeno-associated viruses, Herpes virus, mumps virus, poliovirus, lentivirus, retroviruses, Sindbis virus, and vaccinia viruses such as avian pox virus. The DNA can be introduced into cells in the form of a linear, double stranded (with or without single stranded regions at both ends), single stranded or circular molecules. When the construct is introduced into host cells in the form of two separate DNA fragments, both fragments share some sequence homology

(Overlapping) at the 3 'end of one fragment and at the 5' end the fragments of the other, wherein one of the fragments carries the first targeting sequence while the other fragment carries the other targeting sequence. Upon introduction into the cell, homologous recombination can take place between the two fragments to form a single molecule carrying both the first and second targeting sequences, which delimit the overlap region between the two original fragments. The resulting molecule is then in a form capable of homologous recombination with target sites in the cellular genome. More fragments than the two can be used, each of which is designed such that homologous recombination occurs between the fragments, the only product being a DNA fragment capable of homologous recombination with the target sites in the cell as described above.

If the DNA construct of the invention does not carry a selection marker per se, it may be co-transfected or co-infected with another construct carrying such a trait. The targeting plasmid may be cleaved with a restriction enzyme at one or more sites prior to transfection or infection to form a linear or disrupted molecule. The resulting free DNA ends increase the frequency of homologous recombination. In addition, the free ends of the DNA can be treated with exonuclease to produce overlapping 3 'or 5' single-stranded DNA ends (e.g. at least 30 nucleotides in length, preferably 100-1000 nucleotides in length). These ends again increase the frequency of homologous recombination desired. homologous recombination between the targeting sequence and its target sequence in the genome two copies of the targeting sequences that flank the elements contained in the introduced plasmids.

Cells can be transfected with DNA constructs (preferably in vitro) by a variety of physical or chemical methods including, for example, electroporation, microinjection, microprojectile bombardment, calcium phosphate precipitation, liposome transfection, or DEAE-dextran mediated transfection.

Transfected or infected cells are cultured under conditions that allow homologous recombination to occur as described in the literature (see, eg, Capecchi, Science 24: 1288-1292, 1989). When homologously recombinant cells are cultured under conditions permitting transcription of DNA, the regulatory region introduced by the DNA construct affects transcription of the IFNA2 gene.

Homologously recombinant cells (i.e., cells in which the desired homologous recombination has taken place) can be identified by assessing their phenotype or by analyzing the supernatant of cell culture, where the presence of IFNA2 in the medium is determined by ELISA (enzyme immunoassay). Commercial ELISA kits for the IFNA2 assay are available from Biosource International (CAmarillo, CA). Homologously recombinant cells can also be identified by Southern or Northern analysis or by PCR (polymerase chain reaction) method.

As used herein, the term "primary cells" refers to (i) cells present in a suspension of cells isolated directly from vertebrate tissue (prior to sowing, i.e. before binding to a tissue culture substrate such as a petri dish or culture flask); ii) cells present in the culture directly derived from the tissue, iii) cells seeded in the culture for the first time, and iv) cell suspensions derived from these seeded cells. The primary cells may also be cells as naturally occurring in the human or animal body.

Secondary cells are cells in all subsequent cultivation steps. That is, since the seeded primary cells are first separated from the culture substrate and replanted (passaged), they are referred to herein as secondary cells. Likewise, all subsequent passages are referred to as secondary. Secondary cell strains consist of secondary cells that have been passaged one or more times. Secondary cells typically exhibit a finite number of average culture doublings and contact inhibition of growth and substrate-dependent growth (substrate-dependent growth does not occur in cells that are cultured in suspension culture). Neither primary nor secondary cells are immortal.

- 12GB 299418 B6

Immortal cells are cell lines (as opposed to cell strains where the term "strain is reserved for primary and secondary cells") that appear to exhibit unrestricted growth in culture.

Cells selected for transfection or infection can fall into four types or categories: (i) cells that do not, or do not contain more than a trace amount of IFNA2 protein, (ii) cells that make or contain a protein, but in a quantity other than desired (e.g. (iii) cells that make up the protein in an amount that is physiologically normal for that cell type, but for which it is desirable to extend or increase protein production, and (iv) cells, which are desirable to alter the nature of protein production or to regulate the induction of the gene encoding the protein.

Primary, secondary and immortal cells selected for transfection or infection by the proposed methods can be obtained from a variety of tissues and include all relevant cell types that can be grown in culture. Suitable primary and secondary cells include, for example, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, nerve cells, blood elements (e.g., lymphocytes, bone marrow cells), muscle cells and precursors of these somatic cell types. If we intend to use homologously recombinant cells in gene therapy, the primary cells of the most recent 20 are obtained from an individual to which the transfected or infected primary or secondary cells will be administered. However, primary cells can also be obtained from a donor (i.e., a non-recipient individual) of the same species.

Suitable examples of immortalized cell lines suitable for protein production or gene therapy include, but are not limited to, ovarian cancer cells 2780AD (Van der Blick et al., Cancer. Res. 48: 5927-5932, 1988), A549 (American). Type Culture Collection ("ATCC") CCL 185), BeWo (ATCC CCL 98), Bowes melanoma cell (ATCC CRL 9607), CCRF-CEM (ATCC CCL 119), CCRF-HSB-2 (ATCC CCL 120.1), COLO201 ( COLCC20L (ATCC CCL 222), COLO 320DM (ATCC CCL 220), COLO 320HSR (ATCC)

CCL 220.1), Daudi cells (ATCC CCL 213), Detroit 562 (ATCC CCL 138), HeLa cells and their derivatives (ATCC CCL 2, 2.1 and 2.2), HTC 116 (ATCC CCL 247), HL-60 (ATCC CCL 240) HT 1080 (ATCC CCL 121), 1MR-32 (ATCC CCL 127). Jurkat cells (ATCC TIB 152), K-562 leukemia cells (ATCC CCL 243), KB carcinoma cells (ATCC CCL 17), KG-1 (ATCC CCL 246), KG-1α (ATCC CCL 246.1), LSI23 (ATCC CCL) 255), LS174T (ATCC CCL

CL-188), LSI80 (ATCC CCL CL-187), MCF-7 breast cancer cells (ATCC BTH 22), MOLT-4 cells (ATCC CRL 1582), Namalwa cells (ATCC CRL 1432), NCI-H498 (ATCC CCL) 254); NCI-H508 (ATCC CCL 253); NCI-H548 (ATCC CCL 249); NCI-H716 (ATCC CCL 251); NCI-H747 (ATCC CCL 252); NCI-H1688 (ATCC CCL 257); H2126 (ATCC CCL 256), Paradise cells (ATCC CCL 86), RD (ATCC CCL 136), RPMI 2650 (ATCC CCL 30), RPMI

8226 (ATCC CCL 155), SNU-C2B (ATCC CCL 250.1), SNU-C2B (ATCC CCL 250), SW-13 (ATCC CCL 105), SW48 (ATCC CCL 231), SW403 (ATCC CCL230), SW480 (ATCC) CCL 227), SW637 (ATCC CCL 235), SW937 (ATCC CCL 237), SW1417 (ATCC CCL 238), SW1463 (ATCC CCL 234), T84 (ATCC CCL 234) 248), U-937 cells (ATCC CRL 1593), WiDr (ATCC CCL 218), WI-38VA13 derived line

2R4 (ATCC CCL 75.1); heterohybridoma cells prepared by fusion of human cells and cells of other species. In addition, secondary human fibroblast cells such as WI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171) may be used. In addition, other primary, secondary or immortal cells of human or other animal species may be used for in vitro protein production as well as gene therapy.

Cells expressing IFNA2.

Homologously recombinant cells of the present invention express IFNA2 in the desired amount and are suitable for both in vitro and gene therapy IFNA2 production.

- 13 GB 299418 B6

Protein production.

Homologously recombinant cells of the present invention can be used to produce IFNA2 in vitro. The cells are cultured under conditions that, as described in the literature, result in protein expression. The IFNA2 protein may then be purified from either cell lysates or cell supernatants. A pharmaceutical composition comprising the IFNA2 protein can be delivered to a human or animal organism by methods conventional in pharmacy and known in the art (e.g., orally, intravenously, intramuscularly, intranasally, pulmonary, transmucosal, intradermally, rectally, intrathecally, transdermally, subcutaneously, intraperi10 tonal or intralesional). way). Oral administration may require the use of a strategy to protect the protein from its degradation in the gastrointestinal tract, for example by encapsulation in polymeric microcapsules.

Gene therapy.

The homologously recombinant cells of the present invention are suitable for use as a population of homologously recombinant cell lines, as a population of homologously recombinant primary or secondary cells, as a population of homologously recombinant cell clones or lines, as homologously recombinant heterogeneous cell strains or lines and as cell mixtures in which at least one representative cell of one of the four preceding categories of homologously recombinant cells present. Such cells may be used in a suitable system and administration route to treat (i) infections caused by viruses such as papilloma virus, hepatitis B and C viruses, vaccinia virus, herpes simplex virus, herpes zoster varicellosus virus and rhinovirus, and (ii) any other conditions that can be treated with IFNA2.

Homologously recombinant primary cells, clonal cell strains, or heterologous cell strains are administered to an individual whose abnormal or undesirable condition requires sufficient treatment and prevention and adequately administered expressed or otherwise accessed protein or exogenous DNA in a physiologically relevant amount. A physiologically relevant amount is one that either approaches the normal level of protein production in the body or is increased due to an abnormal or undesirable condition. If the cells are syngeneic to the immunocompetent recipient, they may be administered or implanted intravenously, intraarterially, subcutaneously, intraperitoneally, intraomentally, subrenally capsular, intrathecal, intracranial, or intramuscular.

If the cells are not syngeneic and the recipient is immunocompetent, the homologously recombinant cells may be enclosed in a composition with one or more semipermeable barriers prior to administration. The permeability of the composition is such that the cells cannot leave the composition in which they are administered after implantation, but for the therapeutic protein the barrier is freely permeable and the pro40 tein may diffuse into the space surrounding the implant or enter the systemic circulation. See, for example, U.S. Patent No. 5,641,670; 5,470,731; 5,620,883; U.S. Pat. No. 5,487,737 and co-owned US patent application entitled "Delivery of Therapeutic Proteins" (Applicants: Justin C. Lamsa and Douglas A Treco), April 16, 1999; all of which are incorporated herein by reference. The barrier device may be implanted at any suitable location:

for example, intraperitoneally, intrathecally, subcutaneously, intramuscularly, in a renal capsule or in the omentum.

Barrier formulations are particularly useful and allow the implantation of homologously recombinant immortal cells, homologously recombinant cells from other species (homologously recombinant xenogeneic cells), or cells derived from a donor that is not histocompatible with the acceptor (homologously recombinant allogeneic cells) for treatment of the subject. These compositions maintain the cells in a fixed position in vivo and protect the cells from the host immune system. Barrier formulations also allow for convenient short-term (transient) therapy, as they allow rapid cell removal when, for some reason, the treatment regimen needs to be discontinued. Transfected or infected xenogenic and allogeneic

- 14GB 299418 B6 cells may be used for short-term gene therapy without a barrier device. In such a case, IFNA2 produced by the cells is released in vivo until the cells are rejected by the host immune system.

A variety of synthetic, semisynthetic or natural filter membranes may be used for this purpose including, but not limited to, cellulose, cellulose acetate, nitrocellulose, polysulfone, polyvinylidene difluoride, polyvinyl chloride polymers and polyvinyl chloride derivative polymers. Barrier compositions allow the use of primary, secondary, or immortal cells in gene therapy in humans.

Another type of device for use in the gene therapy of the invention is an implantable collagen matrix in which the cells are anchored. Such a composition, which may comprise beads to which cells are bound, is described in WO 97/15 195, which is incorporated herein by reference, which composition may be implanted as described above.

The amount of cells required for appropriate dose or implantation depends on a number of factors including the level of protein expression, the size and condition of the host animal, and the various limitations associated with the implantation process. Typically, the amount of cells implanted in an adult human or other similar sized animal is in the range of 1 x 10 ⁴ to 5 x 10 ¹⁰ , preferably 1 x 10 ^4.

10 ⁸ to 1 x 10 ⁹ . If necessary, patient cells may be implanted at multiple sites, either once or repeatedly over months or years. The dose may be repeated as necessary.

Deposit.

According to the Budapest Agreement on the International Recognition of Deposits of Microorganisms for Patent Purposes, plasmid pH2AB was deposited with the American Type Culture Collection (ATCC), Rockville, MD, May 12, 1998. The plasmid is available under Accession Code 209872.

Applicant's representative, Transkaryotic Therapies, Inc. guarantees that the ATCC is a depositary, ensuring the consistency of the deposit and, in the case of granting a patent, its easy accessibility to the public. Any restrictions on the availability of the material to the public will be irrevocably lifted when the patent is granted. The material will be available during the patent procedure to persons entitled to this member of the Commission under 37 CFR 1.14 and 35 U.S.C. §122. The deposited material shall be maintained with all due care necessary to maintain the durability of the material and to prevent its contamination for at least five years from the last request for a sample of the deposited material and in any event for at least thirty (30) years from the date of deposit or patent (whichever is the longest). The representative of the applicant acknowledges his obligation40 to replace the deposit in the event that the depositary (ATCC) is unable to provide a sample due to the on-demand deposit status.

Other embodiments.

It should be noted that although the invention has been explained and exemplified by the detailed description, the foregoing description of the invention is illustrative only and in no way limits the scope of the present invention. This is defined by the scope of the following claims.

Other aspects, advantages and modifications are set forth in the following claims.

- 15GB 299418 B6

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the published sequence (SEQ ID NO: 1) coding regions of the human IFNA2 gene and adjacent 5 'and 3' non-coding sequences (GenBank HUMIFNAA). Sequence of PCR primers

IFN1, IFN2, IFN6 and IFN7 are indicated by arrows.

Figure 2 is a schematic diagram depicting the human genomic region of IFNA2 as an insert in plasmid pA2HB.

Figure 3 shows the nucleotide sequence (SEQ ID NO: 7) of the region upstream of the coding sequence of the human IFNA2 gene. This nucleotide sequence has not been previously published.

Figure 4 shows the nucleotide sequence (SEQ ID NO: 9) of the coding region of human

IFNA2 gene and adjacent 5 'and 3' non-coding sequences. The underlined sequence (SEQ ID NO: 8) has not been published yet. Further shown is a polypeptide sequence (SEQ ID NO: 2) encoded by said gene. The N-terminus of the mature polypeptide is designated "mature".

Figure 5 is a schematic diagram of a construct of the invention. The construct comprises a first targeting sequence (1); an amplifiable feature (AM); a selectable character (SM); a regulatory sequence; CAP instead; exon; splice donor site (SD); intron; a splice acceptor site (SA) and a second targeting sequence (2). The black rectangles indicate the coding DNA and the dotted rectangles indicate the transcribed but non-translated regions.

Figure 6 shows the sequence (SEQ ID NO: 10) in the 5 'direction of the coding sequence of the human IFNA2 gene, including part of the coding sequence. The underlined sequence has been previously published, while the other sequences (4074 to -511 of SEQ ID NO: 11) are new. The sequence at SEQ ID NO: 12 is framed. The sequence at position 5 'to SEQ ID NO: 12 is SEQ ID NO: 7. The sequence between the boxed region and the underlined sequence is SEQ ID NO: 8. Nucleotides

-4074 to -3270 is designated as SEQ ID NO: 15. Nucleotides -3267 to -3239 are referred to as

SEQ ID NO: 16. Nucleotides-3241 to 3137 are referred to as SEQ ID NO: 17. Nucleotides-3139 to -511 are referred to as SEQ ID NO: 18.

Figure 7 shows the first targeting sequence (SEQ ID NO: 13) used in the construct of the invention.

Figure 8 shows the second targeting sequence (SEQ ID NO: 14) used in the construct of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Protein production.

Homologously recombinant cells of the present invention can be used to produce IFNA2 in vitro. The cells are cultured under conditions that, as described in the literature, result in protein expression. The IFNA2 protein may then be purified from either cell lysates or cell supernatants. A pharmaceutical composition comprising a protein

IFNA2 can be delivered to a human or animal organism by methods conventional in pharmacy and known in the art (for example, orally, intravenously, intramuscularly, intranasally, pulmonary, transmucosal, intradermally, rectally, intrathecally, transdermally, subcutaneously, intraperitoneally or intralesionally). Oral administration may require the use of a strategy to protect the protein from its degradation in the gastrointestinal tract, for example by encapsulation in polymeric microcapsules.

-16GB 299418 B6

Example 2: Gene Therapy.

The homologously recombinant cells of the present invention are suitable for use as a population of homologously recombinant cell lines, as a population of homologously recombinant primary or secondary cells, as a population of homologously recombinant cell clones or lines, as homologously recombinant heterogeneous cell strains or lines and as cell mixtures in which at least one representative cell of one of the four preceding categories of homologously recombinant cells is present. Such cells may be used in a suitable system and administration route to treat (i) infections caused by viruses such as papilloma virus, hepatitis B and C viruses, vaccinia virus, herpes simplex virus, herpes zoster varicellosus virus and rhinovirus and (ii) ) any other conditions that can be treated with IFNA2.

Example 3:

Another type of device for use in the gene therapy of the invention is an implantable collagen matrix in which the cells are anchored. Such a composition, which may comprise beads to which cells are bound, is described in WO 97/15 195, which is incorporated herein by reference, which composition may be implanted as described above.

The amount of cells required for appropriate dose or implantation depends on a number of factors including the level of protein expression, the size and condition of the host animal, and the various limitations associated with the implantation process. Typically, the amount of cells implanted in an adult human or other similar sized animal is in the range of 1 x 10 ⁴ to 5 x 10 ¹⁰ , preferably 1 x 10 ^4.

10 ⁸ to 1 x 10 ⁹ . If necessary, patient cells may be implanted at multiple sites, either once or repeatedly over months or years. The dose may be repeated as needed.

Claims (38)

PATENT CLAIMS 35 1. DNA construct affecting, after integration into mammalian cell genome via homologous recombination, expression of endogenous interferon-α gene 2 in such cells comprising (i) a targeting sequence comprising at least 20 consecutive nucleotides of SEQ ID NO: 11 and (ii) a transcriptional regulatory sequence. The DNA construct of claim 1, wherein said construct further comprises an exon, a splice donor site, an intron, and a splice acceptor site. 3. The DNA construct of claim 1, wherein said construct further comprises a selection marker. 4. The DNA construct of claim 1, wherein said targeting sequence comprises at least 50 consecutive nucleotides of SEQ ID NO: 11. 5. DNA construct affecting, after integration into the genome of the cell via homologous recombination, 50 expressing an endogenous interferon-α2 gene in mammalian cells comprising (i) a targeting sequence comprising at least 20 consecutive nucleotides of SEQ ID NO: 7 and (ii) a transcriptional regulatory sequence. 6. The DNA construct of claim 5, wherein said construct is further 55 includes an exon, a splice donor site, an intron, and a splice acceptor site. - 17GB 299418 B6 7. A DNA construct affecting, after integration into the genome of a cell via homologous recombination, the expression of an endogenous interferon-α2 gene in mammalian cells, characterized in that it comprises (i) a targeting sequence comprising at least 20 consecutive nucleotides of SEQ ID NO. 5 NO: 8; and (ii) a transcriptional regulatory sequence. 8. The DNA construct of claim 7, wherein said construct further comprises an exon, a splice donor site, an intron, and a splice acceptor site. ío An isolated nucleic acid comprising at least 50 contiguous nucleotides of SEQ ID NO: 11 or a sequence complementary to SEQ ID NO: 11, wherein said isolated nucleic acid does not encode the full-length interferon-α 2. 10. An isolated nucleic acid of claim 9 comprising 15 of at least 100 consecutive nucleotides of SEQ ID NO: 11 or a sequence complementary to SEQ ID NO: 11. 11. The isolated nucleic acid of claim 9 comprising at least 200 contiguous nucleotides of SEQ ID NO: 11 or the sequence of SEQ ID NO: 11. 20 complementary. 12. The isolated nucleic acid of claim 9, wherein SEQ ID NO: 7 or the sequence of SEQ ID NO: 7 is complementary. that it contains The isolated nucleic acid of claim 9, characterized by SEQ ID NO: 8 or a sequence complementary to SEQ ID NO: 8. by including An isolated nucleic acid according to claim 9, characterized by SEQ ID NO: 11 or a sequence complementary to SEQ ID NO: 11. by containing 15. The isolated nucleic acid of claim 9, comprising at least 20 consecutive nucleotides of SEQ ID NO: 7 or a sequence complementary to SEQ ID NO: 7. 35 16. The isolated nucleic acid of claim 9 comprising at least 20 consecutive nucleotides of SEQ ID NO: 8 or a sequence complementary to SEQ ID NO: 8. 17. An isolated nucleic acid comprising a chain comprising A 40 nucleotide sequence that (i) is at least 200 nucleotides in length and (ii) hybridizes under high stringency conditions to SEQ ID NO: 11 or a sequence that is complementary to SEQ ID NO: 11. 18. The isolated nucleic acid of claim 17, wherein said nucleic acid is as follows The 45 nucleotide sequence is at least 400 nucleotides long. 19. The isolated nucleic acid of claim 17, wherein said nucleotide sequence is at least 1000 nucleotides in length. 50 20. An isolated nucleic acid comprising a strand comprising a nucleotide sequence that (i) is at least 100 nucleotides in length and (ii) hybridizes under high stringency conditions to SEQ ID NO: 7 or a sequence that is to SEQ ID NO. NO: 7 complementary. - 18GB 299418 B6 21. An isolated nucleic acid comprising a strand comprising a nucleotide sequence that (i) is at least 50 nucleotides in length and (ii) hybridizes under high stringency conditions to SEQ ID NO: 8 or a sequence that is to SEQ ID NO. NO: 8 complementary. 5 22. An isolated nucleic acid comprising a strand comprising a nucleotide sequence which (i) is at least 50 nucleotides in length and (ii) has at least 80% identity to a fragment of SEQ ID NO: 11 of the same length as said nucleotide sequence . 23. The isolated nucleic acid of claim 22, wherein said 10 nucleotide sequence is at least 100 nucleotides in length. 24. The isolated nucleic acid of claim 22, wherein said fragment forms part or all of SEQ ID NO: 7. 15 Dec 25. The isolated nucleic acid of claim 22, wherein said fragment forms part or all of SEQ ID NO: 8. 26. A homologously recombinant mammalian cell stably transfected with a DNA construct according to claim 1, characterized in that it has undergone homologous recombination between the construct 20 and the genomic DNA upstream of the endogenous ATG initiation codon, in one or both alleles of the endogenous. IFNA2 gene. 27. A homologously recombinant mammalian cell stably transfected with a DNA construct according to claim 2, characterized in that it has undergone homologous recombination between the const25 and the genomic DNA upstream of the endogenous ATG initiation codon, in one or both alleles of the endogenous. IFNA2 gene. 28. A homologously recombinant mammalian cell stably transfected with a DNA construct according to claim 3, characterized in that it has undergone homologous recombination between the construct and the genomic DNA at a site upstream of the endogenous ATG initiation codon, in one or both alleles of the endogenous. IFNA2 gene. 29. A homologously recombinant mammalian cell stably transfected with a DNA construct according to claim 4, wherein the homologous recombination has occurred between the construct and the genomic DNA upstream of the endogenous ATG initiation codon, in one or both alleles of the endogenous IFNA2. gene. 30. A method for affecting the expression of an endogenous interferon-alpha2 gene in a mammalian cell in vitro, comprising introducing the DNA construct of claim 1 into the cell. 40, and maintaining cells under conditions that allow for homologous recombination between the construct and the target site located 5 'to the coding sequence of the endogenous interferon-α 2 gene. Use of a cell according to claim 26 for the preparation of a medicament for the delivery of interferon-α 2, 45 wherein the cell secretes interferon-α 2. Use of a cell according to claim 27 for the preparation of a medicament for the delivery of interferon-α 2, wherein the cell secretes interferon-α 2. 50 Use of a cell according to claim 28 for the preparation of a medicament for the delivery of interferon-α 2, wherein the cell secretes interferon-α 2. The use of a cell according to claim 29 for the preparation of a medicament for the delivery of interferon-α 2, wherein the cell secretes interferon-α 2. - 19GB 299418 B6 35. A method of producing interferon-α2, comprising preparing the cells of claim 26, and further culturing said cells in vitro under conditions that allow the cells to express and secrete interferon-α2. 5 36. A method of producing interferon-alpha 2 comprising preparing cells according to claim 27 and further culturing said cells in vitro under conditions that allow the cells to express and secrete interferon-alpha 2. 37. A method of producing interferon-alpha 2 comprising preparing the cells of claim 28 and culturing said cells in vitro under conditions that allow the cells to express and secrete interferon-alpha 2. 38. A method of producing interferon-alpha 2 comprising preparing cells according to claim 29 and further culturing said cells in vitro under conditions that allow the cells to express and secrete interferon-alpha 2.