WO1996012804A2

WO1996012804A2 - Recombinant dna

Info

Publication number: WO1996012804A2
Application number: PCT/GB1995/002520
Authority: WO
Inventors: Nicholas Yannoutsos
Original assignee: Imutran Limited
Priority date: 1994-10-25
Filing date: 1995-10-25
Publication date: 1996-05-02
Also published as: IL115727A0; ZA959051B; CA2202130A1; GB9421464D0; JPH10508196A; NZ294293A; EP0788544A2; WO1996012804A3; AU3705495A

Abstract

The complete human CD59 gene has been cloned. The gene is useful in the preparation of transgenic xenograft donor animals. As part of the cloning procedure, concentration and purification of a crude DNA preparation including high concentrations of spermine and spermidine was carried out by dialysis.

Description

REC0M3INANT DNA

This invention relates to the cloning of CD59, particularly human CD59.

The lysis of cells by complement requires only the terminal components C5, C6, C7, C8 and C9 and is initiated by the cleavage of C5 to C5b. Sequential addition of C6, C7, C8 and C9 to C5b leads to the formation of the membrane attack complex (MAC) which, when inserted into the lipid bilayer, can form transmembrane pores. It is well known that when complement of one species is activated on homologous erythrocytes, lysis is much less efficient than when it is activated on other species of cell. The down- regulation of complement-mediated lysis of homologous cells is known to be the result of various homologous complement restriction factors (HCRFs) , of which decay accelerating factor (DAF) and MAC-inhibiting protein (MIP) were two of the first identified members. Davies et al . (J. Exp . Meά . , 170 637-654 (1989)) reported the identification of a 20 kDa membrane protein which is also capable of inhibiting complemen -mediated cell lysis (and which is therefore a further HCRF) . The protein was designated CD59 at the 4th Leucocyte Workshop.

Subsequent work has shown that CD59 is a structural analogue of murine Ly-6 antigens (Philbrick et al . , Eur. J. Immunol . 20 87-92 (1990)) and that its homologous complement restriction activity is mediated by a species- selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9 (Rollins et al . , J. Immunol . 7 2345-2351 (1991) . DNA sequences encoding homologous complement restriction factors such as DAF, MIP and, now, CD59 are of particular interest because animals which are transgenic for HCRFs of a given species may be expected to be suitable xenotransplantation donors for that species even though the transgenic animal is itself a species which is discordantly related to the graft recipient species; this pioneering work is disclosed in WO-A-9105855. In fact, Walsh et al . {Eur. J. Immunol . 21 847-850 (1991)) have confirmed that transfection of CD59 cDNA into rat cells confers resistance to human complement. In this way, therefore, pigs which are transgenic for human CD59 or another HCRF may be suitable donors of kidneys and other organs for xenografting into human recipients.

Davies et al . supra cloned a cDNA encoding CD59 from a human T-cell cDNA library in COS cells. Brinster et al . ( Proc . Na t ' l . Acad . Sci . USA, 85, 836-840 (1988)) have demonstrated, though, that introns increase the transcriptional efficiency of transgenes, at least in transgenic mice. There has therefore been considerable interest in seeking to provide a genomic clone, as opposed to a cDNA clone, of human CD59.

Although the CD59-encoding gene has attracted some attention (Tone et al . , JM Mol . Biol . , 2217, 971-976 (1992) and Petranka et al . , Proc . Na t ' l . Acad . Sci . USA, 89, 7876-7879 (1982)), the entire human CD59 gene has so far been refractory to successful cloning attempts and, indeed, there is confusion in the literature on the position of the first exon. Tone et al . , for example, speculate that the first exon is about 30 kb upstream

(that is, to the 5' -side) of the rest of the gene.

Conversely, Petranka et al . give an alternative map of the CD59-encoding gene, which would have the first exon closer to the rest of the gene.

The present invention has overcome the difficulties in the art and enables the provision of the entire human CD59 gene, and related molecules, in a useful form.

According to a first aspect of the invention, there is provided a recombinant or isolated DNA molecule comprising a first series of sequences which together encode a protein having CD59 activity and a second series of sequences which are identical or substantially identical to the introns which naturally occur in a CD59 gene, members of the second series of sequences being distributed among members of the first series.

Members of the first series of sequences may be identical to exons in the natural (particularly human) CD59 gene. However, complete identity to a wild-type consensus sequence is not essential; natural or artificial mutants, variants and derivatives are also within the scope of the invention. In particular, soluble CD59 (in which the membrane anchoring domain is absent) may be encoded by DNA molecules of the invention. The sequence coding for the missing domain of CD59 may be physically or functionally absent in the DNA sequence. Functional absence may be achieved by inserting one or more stop codons before the appropriate sequence or by other suitable manipulatory techniques. The invention also comprehends sequences encoding modifications of the natural CD59 molecule which retain the homologous complement restriction factor activity of CD59; examples include modified molecules having increased or other modified activity or resistance to degradation (for example in a transgenic host or xenogenic graft recipient) or other advantageous properties.

The second series of seσuences may be identical to the introns occurring in a natural (particularly human) CD59 gene, which may be a human CD59 gene. However, complete identity is not essential. The invention only has as its requirement substantial identity. "Substantial" is to be understood functionally, in other words sufficient identity is needed to give the enhanced level of expression (or better than the enhanced level of expression) normally seen by the complete natural gene over the corresponding cDNA sequence. Generally, there will be at least 90 or even 95% homology with natural intron sequences. Usually, all the natural introns (or sequences substantially __identical to them) will be present, but this is not necessarily so; for example, if one part of the natural CD59 molecule is to be omitted and exons encoding that part (an intron) , then that intron may be omitted. At least the first intron will generally be present.

Distributing of the second series of sequences among the first series of sequences may be the same as the natural intron/exon distribution. Again, this is not essential and in some cases, a non-natural distribution may be acceptable or even preferred.

DNA molecules in accordance with the invention may include CD59 flanking sequences, which may have originated from locations upstream and/or downstream of the CD59 gene. Such flanking sequences may contain important regulatory sequences; for example, the natural promoter will be located upstream of the gene itself. It is preferred, in the practice of the present invention, for upstream of downstream extra sequences to be identical, or substantially identical, to natural CD59 flanking sequences. At least 10, 50, 100, 200 or 250 kb upstream and/or downstream may be included. If both 250 kb upstream and 250 kb downstream are included, the overall maximum size of the DNA molecule in accordance with the invention is likely to be about 550 kb, given that the size of the complete CD59 gene is about 50 kb.

A recombinant DNA molecule in accordance with the invention may be in the form of a vector. Vectors may include sequences facilitating cloning and selection and may (but do not have to) contain sequences (such as a promoter) enabling expression of the encoded CD59. A recombinant DNA molecule in accordance with the invention may alternatively or additionally be in the form of a construct suitable_for preparing a transgenic animal, for example by microinjection.

In a second aspect, the invention provides a process for preparing DNA as described above, the process comprising coupling together successive nucleotides and/or ligating oligo- or poly-nucleotides. Once a template molecule has been prepared, further DNA molecules in accordance with the invention will generally be prepared by DNA replication, usually in vivo.

In accordance with a third aspect of the invention, there is provided a host comprising a DNA molecule as described above, for example as heterologous DNA. The host may be a unicellular organism. In such a case, the organism may be prokaryotic (for example E. coli ) or eukaryotic (such as yeast) . Alternatively, the host may be a cell line of any suitable animal (particularly mammalian) species. Most preferred of all as a host in accordance with the invention is a transgenic animal containing a transgene including a DNA sequence as described above; such transgenic animals may be prepared as described in, and/or by the techniques referred to in, WO-A-9105855. By virtue of the transgene, the transgenic animal should be capable of expressing CD59 on at least some of its organs, to enable those organs to be xenografted into human recipients. The animal may even be discordantly related to humans, and may be a pig.

In the making of the invention, the complete human CD59 gene was successfully obtained by screening a human YAC (yeast artificial chromosome) library. Such libraries are publicly available for screening, as detailed in the examples below. As a screening probe, CD59 cDNA was used. Although the cDNA was itself recloned for this purpose, the cDNA of Davies et al . supra could have been used without significant modification.

The medium against which the DNA preparation is dialysed will be any suitable medium, such as those routinely used in the art. Typically, the medium may contain 0.1 M NaCl, 10 mM Tris.HCl pH8 and 0.1 mM EDTA pH8. The medium may contain a significant concentration (for example about 20%) of polyethylene glycol or any other large macromolecule unable significantly to pass through the dialysis membrane.

Dialysis may be carried out through any suitable membrane having regard to the size of the DNA. The membrane may be formed as a bag, but it does not have to be. Collodion bags are the most preferred forms of dialysis membrane, particularly those having a 75 kDa pore size. An example is that sold by Schleicher & Schuell, Inc. (Keene, New Hampshire, USA) under the trade mark and product designation ULTRA THIMBLE^™ UH 100/75. Dialysis may be assisted by applying a vacuum, if desired, but that is not essential.

In a preferred protocol, dialysis is first carried out under vacuum, in the presence of polyethylene glycol, and secondly not under vacuum, in the absence of polyethylene glycol.

In an important embodiment of the invention, the DNA purified and concentrated as described above encodes human CD59 and comprises a substantially complete copy of the human gene.

Preferred features_of each aspect of the invention are as for each other aspect, mutatis mutandis .

The following examples illustrate the invention.

Individual techniques and protocols, where not spelled out in detail or referenced in specific publications, may be found in Sambrook et al. , "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory Press,

1989.

The examples refer to the accompanying drawing, in which:

FIGURE 1 is a restriction map of a genomic clone of the CD59 gene. EXAMPLE 1 - Screening a human genomic library In view of the large size of the CD59 gene, a human YAC library was screened. YACs are yeast artificial chromosomes, whose structures and properties are reviewed in:

"Guide to Yeast Genetics and Molecular Biology", Guthrie and Fink, Eds., Methods in Enzymology, Academic Press, 1991, Chapter 17; and

"Techniques for the Analysis of Complex Genomes", Anand, Ed., Academic Press, 1992 (Chapters 4,7 and 8 in particular) .

Two papers dealing with specific techniques involving YACs are Gnierke et al . "Microinjection of Intact 200 to 500 kb Fragments of YAC DNA into Mammalian Cells" Genomics 15 659-667 (1993) and Schedl et al. "A Yeast Artificial Chromosome Covering the Tyrosinase Gene Confers Copy Number-Dependent Expression in Transgenic Mice" Nature 326 258-261 (1993) .

In the present example, the human YAC Libraries Nos. 900, 901 and 905 of The Imperial Cancer Research Fund reference library database were screened. Library No. 900 (4X YAC) was constructed from digests of DNA from 4X cell line GM1416B ligated into pYAC4; Saccharomyceε cerevisiae AB1380 was used as host. Library No. 901 (4Y YAC) was constructed from digests of DNA from 4Y cell line OXEN 49XYYYY, ligated into pYAC . S . cerevisiae AB1380 was again the host. Huntington Disease Library No. 905 (HDD was constructed from digests of DNA from lymphoblastoid cell line HD1, ligated into pYAC4. S. cerevisiae AB1380 was again the host. These three human genomic libraries are available for screening at the Reference Library DataBase of The Imperial Cancer Research Fund (Genome Analysis Laboratory, PO Box No 123, London WC2A 3PX) .

Essentially, the libraries were screened with CD59 cDNA as published by Davies et al. A partial cDNA, covering only the protein-coding region of CD59 cDNA was constructed by making primers from Figure 8 of Davies et al . (page 647) both upstream and downstream of the protein-coding region. The polymerase chain reaction was used under standard conditions, using the following primers:

5' side: 5' -CGCAGAAGCGGCTCGAGGCTGGAAGAGGAT-3'

3' side: 5' -AACCTCGAGTTCTCGAGAAGCTCTCCTGGTGTTGAC-3'

to form cDNA from HeLa mRNA. The underlined parts of the above sequences represent Xhol restriction endonuclease recognition sites which were used to subclone the amplified sequence (position 0 to position 490 of the published sequence; the protein sequence is up to position 450) into the expression vector pT7T3 19U (Pharmacia) . Positions refer to the sequence of Davies et al . The PCR was done as follows: 3/xg of HeLa mRNA was used in a 20 μl reverse transcription reaction with 50 pmoles of the 3'-side primer (instead of oligo-dT) and the reaction was directly used in a 100 μl PCR reaction with 50 pmoles of the primers, 1.5 mM MgCl₂ and 30 cycles of 1 minute at 94°C, 1 minute at 52°C and 2 minutes at 72°C.

By this method, cDNA encoding CD59, essentially as described by Davies et al . (J. Exp . Med. , 170 637-654 (1989) ) , was re-cloned. Filters containing clones from all three libraries (900, 901 and 905) were provided by the library laboratory and screened with the above cDNA. Seven positions gave positive signals and their co-ordinates were given to the library laboratory. Two signals were immediately rejected as pseudopositives, since no clones had been plated at these positions on the original grid. The library laboratory sent five stabs with the other clones; they were screened by PCR, using the primers:

5' Side: 5' -GTCAACACCAGGAGAGCTTC-3' 5' -side: 5' -TGCAGAGAACCCACTCAAGC-3'

and the conditions essentially as described above, except that 100 pmoles of each primer was used in a 100 μl reaction, 1.5 mM MgCl₂ and the annealing temperature was 52°C. Southern hybridisation was carried out as in Sambrook et al . One proved to be positive. To guard against impurity, the positive was streaked out on agar and ten clones picked; all, again, proved positive.

EXAMPLE 2 - Isolation of DNA from yeast

The isolation of DNA from yeast needs to be undertaken carefully as yeast DNA can relatively easily fracture during an isolation procedure.

The protocols used for this purpose are derived from the manual of the 6th Wellcome Summer School, DNA Related Methods in Human Genetics: YAC Cloning in Genome Analysis (5 July to 13 July 1991) . In brief, 2% agarose is mixed with an equal volume of cells so that 1% agarose blocks are formed. The wall of the yeast cells is degraded by 1 mg/ml Zymolase and the resulting spheroplasts are destroyed with 2 mg/ml proteinase K in 1% Sarcocyl or with 1% lithium dodecyl sulphate (LDS) . The proteinase K diffused into the plug and digested the cells, thereby releasing DNA in si tu . The resulting agarose piugs were then set into an agarose gel well and subjected to pulse field electrophoresis under the following conditions: pulse 50 seconds - 10 sec in logarithmic ramp (log) , angle 120° - 110° in linear ramp (lin) , voltage 250 - 200 log, rotor speed 5 moving with the power off and temperature at 13°C.

The resulting gel was blotted (like a Southern blot) by capillary action overnight and screened with cDNA, prepared as in Example l, to identify the correct chromosome (the YAC) . As expected, one yeast chromosome band hybridised with the cDNA. Normal yeast chromosomes, which did not contain the human insert, would not be expected to hybridise and—in fact did not do so.

A larger scale (preparative) gel was then prepared and the process essentially repeated, except this time hybridisation with the CD59 cDNA was not carried out. The relevant YAC band had been identified by the smaller scale preparation; to isolate the YAC band from the preparative gel, the two sides of the gel are cut off as two long strips running from the top to the bottom of the gel are stained with ethidium bromide so that all the chromosomes (or, more accurately, the edges of the bands of the c romosomes) are revealed as they have run into the gel. The edges of the YAC band are cut off the stained side strips and the strips are placed on either side of the unstained preparative gel. A long piece of the gel s cut from one side strip to the other at the positions of the side cuts. This piece contains the YAC. This "blind" cutting of the YAC is necessary because staininσ with ethidium bromide and exposure to ultra- violet light will damage the DNA beyond any hope of recovering the YAC intact. (The edges of the band on the side strips are of course not used. )

Because the preparative gel is overloaded, it cannot be run under the same conditions as the small scale, analytical gel. The parameters chosen have to take account of the fact that the gel will run more slowly and have to be optimised to give maximum resolution in the size range around the size of the YAC size (540kb for the genomic CD59 YAC) ; the resolution of the rest of the yeast chromosomes can be completely disregarded. the programme used was, therefore: pulse 40 sec - 10 sec log, angle 135 - 115° log, voltage 180-160 log, rotor speed 3 moving with power off, temperature 10°C for at least 36 hours (60 hours over a weekend is convenient) .

The relevant portion of the gel which had been identified by reference to the earlier, smaller scale preparation, having a 4.5 ml volume, was cut out and mixed with 4.5 ml water and high salt buffer (0.14 M NaCl, final concentration, 40 mM Tris pH 6, 1 mM EDTA) and containing a spermine/spermidine mixture (0.3 mM spermine, 0.75 mM sper idine) . The gel was allowed to equilibrate for 30 minutes.

The gel slice was then cut into three and removed from the liquid. Each of the three pieces fitted into an Eppendorf tube, and the three Eppendorf tubes were put into a 70°C bath for 7 minutes. This melted the agarose.

The Eppendorf tubes were then put into a 40°C bath and allowed to equilibrate for 5 to 10 minutes. The agarose stayed liquid. GELASE agarase (90 units) was added to digest the agarose. After 10 minutes, ten further units of agarase were added, and the mixture was allowed to incubate for a further 30 minutes.

The Eppendorf tubes were then spun for 15 minutes at room temperature at approximately 14,000 g. The undigested agarose formed a pellet, which was discarded. The supernatants of the tubes were pooled (total volume about 4 ml) and put into a collodion bag. Concentration was achieved by vacuum dialysis; dialysis was conducted against:

0.1 M NaCl 10 mM Tris.HCl pH 8.0 0.1 mM EDTA pH 8.0 20% polyethyleneglycol; until the volume is visually estimated to be eight-fold less.

After dialysis, the bag was retrieved and its outside briefly rinsed with distilled water to remove the polyethyleneglycol. The contents were transferred to a fresh container for dialysis without vacuum against the buffer specified above but modified by the omission of the polyethyleneglycol. This second dialysis can be continued for 1 to 2 hours, or longer if care is taken that the dialysis be stopped before the volume starts to become appreciably big again. In the present instance, the volume immediately after the vacuum dialysis was 0.5 ml, and after the second dialysis step it was 0.8 ml.

EXAMPLE 3 - Preparation of transgenic animals

Using the standard Hogan microinjection protocol (Hogan et al . , "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1986)), transgenic animals can be prepared using the final DNA preparation of Example 2 above

EXAMPLE 4 - Characterisation Characterisation of the genomic CD59 clone from the library can be done at the plug step of Example 2. Restriction enzymes can diffuse into the plug, which may therefore simply be dipped into enzyme in appropriate buffer. Restriction mapping can be done conventionally. A restriction map of the clone is shown in Figure 1.

EXAMPLE 5 - Deposit of clone

Cloned genomic DNA resulting from Example 2 was deposited under the terms of the Budapest Treaty at The National Collections of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen, AB2 1RY, Scotland on 12 October 1994 in a yeast ( Saccharomyces cerevisiae AB 1380 CD59 YAC) host and given the accession number NCIMB 40690.

Claims

1. A recombinant or isolated DNA molecule comprising a first series of sequences which together encode a protein having CD59 activity and a second series of sequences which are identical or substantially identical to the introns which naturally occur in a CD59 gene, members of the second series of sequences being distributed among members of the first series.

2. DNA as claimed in claim 1, wherein the members of the first series of sequences comprise all the exons in the human CD59 gene.

3. DNA as claimed in claim 1, wherein the members of the first series of sequences encode soluble CD59.

4. DNA as claimed in claim 1, 2 or 3, wherein the second series of sequences comprises the introns occurring in the human CD59 gene.

5. DNA as claimed in any one of claims 1 to 4, wherein the distribution of the second series of sequences among the first series of sequences corresponds to the natural CD59 genomic intron/exon distribution.

6. DNA as claimed in any one of claims 1 to 5, which comprises CD59 flanking sequences.

7. DNA as claimed in any one of claims 1 to 6 which is in the form of a vector.

8. A process for preparing DNA as claimed in any one of claims l to 7, the process comprising coupling together successive nucleotides and/or ligating oligo- or poly- nucleotides.

9. A host comprising a DNA molecule as claimed in any one of claims 1 to 8.

10. A host as claimed in claim 9, which is a transgenic animal.

11. A method of purifying and/or concentrating a DNA preparation, the method comprising adding spermine and spermidine to the DNA preparation and dialysing the preparation against an appropriate medium, wherein the final spermine concentration is at least 1.5 mM and the final spermidine concentration is at least 0.6 mM.

12. A method as claimed in claim 11, wherein the final spermine concentration is about 4.5 M and the final soermidine concentration is about 1.8 M.

13. A method as claimed in claim 11 or 12, wherein the medium against which the DNA preparation is dialysed contains 0.1 M NaCl, 10 mM Tris.HCl pH8 and 0.1 mM EDTA.