CA2189438A1 - Dna sequences - Google Patents

Abstract

Poor expression yields of recombi-nant human factor IX are attributable to aberrant splicing in heterologous expres-sion systems such as transgenic hosts.
The aberrant splicing sites have been identified as (a) a donor site including mRNA nucleotide 1085; and (b) an ac-ceptor site including mRNA nucleotide 1547; adopting the mRNA nucleotide numbering of Figure 2 of the drawings.
Improved factor IX expression sequences have at least one of these sites engineered out, so as to prevent or reduce the ef-fect of aberrant splicing and to increase yields. The improved DNA sequences may also be useful in gene therapy.

Description

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This invention relates to DNA sequences Pnco~1;ng human factor IX (fIX) . Such sequences are useful in expression 5 systems for factor IX, ;nnlllfl;n~ transgenic animals, and also have potential in gene therapy.
It is difficult to achieve high expression yields of factor IX in heterologous, particularly trAn~ n;c~
systems. For example, while the basic approach to ,~-lArto~lnhul; n-driven tran8genic expression of human factor IX in the milk of transgenic animals such as sheep (as disclosed in WO-A-8800239) does work, the yields obtained are low. There seem to have been two main reasons for this:
Failure to exprell~ . The use of f actor IX cDD~As has generally proved a problem in terms of getting reasonable levels of the ~ Liate fIX transcript.
This problem was partially solved by the ~r~nc~PnP
rescue approach (described in WO-A-9211358, "Increased Expression by a Second Transferred Sequence in Transgenic Organisms " ~ . In this prior publication, co;ntP~--ati~n of ~-lArto~lnbul;n (BLG) with the human factor IX-Pn-nrl;n~ construct FIXD led to the prs~lllnt; nn of li~es of mice expressing high levels of FIXD mRNA. The milk of these animals, however, ,~nr1tA;nP~ very little fIX.
Aberra~t 13p~ic~n5. Closer ;n~pPc~c;nn of the FIXD
mRNA transcripts in the sLGi FIXD mice showed that they were approximately 450 bp shorter than predicted. It was surmised that these are deleted internally most probably by an aberrant splice of Wo ss/3oooo 2 1 8 ~ 4 3 ~

the m'.~NA (Clark et al., Bio/Technology 10 1450-1454 (1992) ) .
Splicing of human factor IX m.~NA in liver cells has been s discussed in J. Biol. Che . 270, 5276-5281 (1994) (Kurachi et al). Here it i8 indicated that the presence of splicing signal sequences results in increased expression of factor IX since qr7;cec~ ~.n~TllPYPC act to protect precursor mRNAs from random degradation before being transported out of the nucleus.
It has now been detPr~ nPr7 that aberrant q~ ;n~ is indeed a cause of low factor IX yield in heterologous or transgenic expression systems. Furthermore, and most significantly, the location of cryptic splice sites in the human gene Pn,-or7;n~ factor IX has been ;r7~nt;f;ed.
This discovery enables factor IX-encoding DNA sequences to be Pn~;nPPred to avoid tae observed aberrant splicing.
According to a first aspect of the present invention, there is provided DNA having a sequence Pn~Qr7; n~ a protein having human factor IX activity, wherein the DNA
is ~ '; f; P~ to interfere with the functioning of at least one of the following cryptic splice sites:
(a) a donor site ;nnll7r7;n~ m.~NA nucleotide 1086;
and (b) an acceptor site including mRNA nucleotide 1547;
adopting the mRNA nucleotide numbering of Figure 2 of the 3 0 drawings .
DNA in a..uLdallce with the invention makes possible much higher levels of fIX expression than hitherto described by correcting an a'.-errant splice of ~IX sequences.

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A donor site in nuclear pre-mRNA (that i8, the primary transcript of the gene which exists transiently in the nucleus before splicing to generate mRNA which is exported to the cytoplasm) rnnt~;n.c the nucleotides GU, which after splicing become the 5~ to~in;ll nucleotides of the excised intron. An acceptor site in the nuclear pre-mRNA rnnt~;nC the nucleotides AG, which after splicing become the 3' torm;n~l nucleotides of the excised intron. ~he nucleotide numbers given in the preceding paragraph are for the G residue of the (5' ) donor site and the G residue of the ~3 ' ) acceptor site, respectively .
Pref erred DNA in accordance with the invention encodes wild-type human factor IX. However, DNA Pnrnri;n~
variants (particularly allelic variants from a cnnconR--c sequence), conservative ~tinn~ or other proteins is also within the scope of the invention, provided that the proteins are subst~nt1Ally i- ~lo~ouc with human factor IX. "Substantial homology~, as is well understood in the art, may be assessed either at the protein level or the nucleic acid level. For example, at the protein level, substantial homology may be said to be present if a r~n~ te protein exhibits amino acid homology to human factor IX at a level of at least 40, 60, 80, 90, 95 or 99~, in increasing order of preference. At the nucleic acid level, substantial homology may be said to be present if a r~nri;fi~te DNA sequence exhibits DNA sequence homology to human factor IX at a level of at least 80, 90, 95 or 99~, in increasing order of preference.
It will be appreciated that the invention has application to a variety of DNA sequences .~nrori;n~ factor IX (or another protein having factor IX activity). In Wo ss/3oooo ~ 1 8 ~ ~ ~ 8 ` ~ 3 ~

particular, the invention is applicable to cDNA
sequences, genomic sPqllPnrPq having a full complement of natural introns and ~'minigene~ sequences, rnnt~;n;nrJ some but not all of the introns present in genomic DNA
Pn~ro~;n~ factor IX.
There are a variety of ways in which DNA in accordance with the invention may be I '; f; P~ to interfere with the fl1nrt;r~n;nr~ of the cryptic donor/acceptor gites 80 as to prevent or at least signif icantly reduce ~h~.rrz~nt splicing .
First, the intron/exon structure of the constructs could be changed, on the basis that additional introns 5~ or 3' would "compete" with the cryptic splice in some way.
owever, this ~ L~a~l may be relatively complex and lead to only partial suppression of aberrant splicing.
Secondly, the cryptic donor site could be Pn~;nPPred out.
Either the G or the U of the mRNA donor site could be replaced with another base, or both could be replaced, provided that a stop codon does not result from the change. This approach is technically simpler than the competitive intron approach described above, but necessitates a change in the amino acid sequence of factor IX, because the GU residues at the donor site form the first two nucleotides of a valine codon, and all valine codons begin GU. This may not be a disadvantage, and may actually be an advantage if a second or sllhse~lPnt generation variant of factor IX is being engineered. ~Iowever, it is not suitable if retention of the wild-type factor IX sequence, at least in the region of the donor site, is PccPnt; ~

wo 9s/30000 2 1 8 ~ ~ 3 8 P~ 9,6 Thirdly, and in most instances preferably, the cryptic acceptor site can be engineered out. This site lies in the 3' untr~ncl~tPd region of factor IX DNA, and 50 there are no implications for the amino acid sequence. Either the A or the G of the mRNA acceptor 6ite could be deleted or replaced with another base, or both could be deleted or replaced. In fact, in some of the simplest f~mho~; -tq of the invention, deletion of the acceptor site j ust requires the production of a f actor IX cI)NA
segment which is shortened at the 3 ' end (or, of course, a DNA other than a cDNA shortened CULL r.Y..,.~;n~1Y). In other ~ ' ~lr ' q, site-directed m~ltagPnpq;q techniques may be used specifically to alter the acceptor site (or, of course, the donor site).
DNA in accordance with the invention is useful in systems for expressing factor IX (or like protein6).
According to a second aspect of the invention, there is provided an expression host comprising DNA in accordance with the first aspect of the invention operably linked to an expression control seallPn~p. The expression control sequence will usually comprise a promoter, and other regulatory sequences may be present.
While the invention may be generally useful across various different cell types and cultured cells, it is with transgenic animal expression systems that the invention has particular application, because of the large yields that are in principle available from this te-~hn~ l Qgy. Therefore, the expression host is in certain f avoured Pmho~l; q an animal, such as a mammal .

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A preferred transgenic system for the production of heterologous proteins involves the use of transgenic E'l~C''nrAl non-human mammals, especially sheep and other dairy animals, which express a transgene in the mammary gland (of an adult female) under t~e control of a milk protein promoter, particularly that of the milk whey protein ,~-lacto~ hlll in, as disclosed in WO-A-8800239, WO-A-9005188 and WO-A-9211385.
However, the iIlve~tion is not limited to the use of these preferred tra~sge~ic systems. It is expected that factor IX-encoding sequences will be used in gene therapy approaches for haemophilia, for example using retroviral vectors or direct transf ection techniques into stem cells . The advantages of an improved f IX sequence which does not aberrantly splice are self evident.
Preferred features for each aspect of the invention are as for each other aspect, mut~tis ~nutandis.
The invention will now be illustrated by the f ollowing examples. The examples refer to the drawings, in which:
FIGTlRE 1 refers to Example 1 and shows the scheme used to confirm the aberrant splicing of the FIXD
construct;
FIG~RE 2 also refers to Example 1 and is adapted from Anson et al., The ~MBO Journai 3 (5) 1053-1060 (1984) and shows the locations of the cryptic donor and acceptor sites in factor IX mRNA;
FIG~RE 3 refers to Example 1 and shows in more detail how the donor and acceptor sites interact;

Wo 9s/30000 2 1 ~ ~ 4 3 8 the f igure also shows generalised consensus sequences for donor and acceptor sites;
FIGURE 4 shows the gross structure of the human factor IX gene, including the locations of the cryptic splice sites;
FIGURE 5 refers to Example 2 and shows a PCR-based scheme for distinguishing between ln~pl i ced and aberrantly spliced mRNA for different constructs and in different expression systems;
FIGURE 6 refers to Example 3 and shows the construction of a construct designated FIXD-~3 ' splice;
FIGURE 7 refers to Example 4 and shows a Western Blotting analysis of milk from tr~nr~n; c mice expressing high yields of human factor IX. Milk samples from two animals from line FIXD~3'-splice (31 31.2 and 31.3) were electrophoresed under non-reducing conditions. Milk samples were diluted 1/200 and either 5 ~Ll or 10 ~1 loaded. fIX, 10 ng fIX; CM, control milk; CM+fIX, control milk + 10 ng fIX; and FIG~7RE 8 also refers to Example 4 and shows Northern blots of representative RNA samE?les from FIXD-~3'splice mice probed with a factor IX-~7pe~;f;c probe. Mammary gland RNAs from high and medium expressing BIX mice ~BIX33.1 and BIX34.1) were compared to mammary gland samples from FIxDa3 ' -splice transgenic mice (labelled BIX~3'3.10-~
BIX~3'44.2) . Blots were probed with l~h~17~ insert WO 95/30000 " 2 1 ~ ~ 4 3 8 P~ . J' . ..)~ ~
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., 8 from p5G3'CVII a plasmid rnnTA;n;nr cDNA sequences human f IX and then reprobed with G~BDH to control for loading. The sizes of the transcripts are indicated. The FIXD~3 ' -splice transcripts are evidently larger than those from the BIX mice.
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The aberrant splicing of the FIXD mRNA was confirmed by cloning these transcripts by RT-PCR from mammary gland RNA of one of the expressing lines of mice. FIXD is disclosed in Example 3 of WO-A-9005188 and Comparative Example 6 of WO-A-9211385 and comprises human factor IX
(fIX) cDNA fused to 3-lactoglrhlll;n (BLG) 5' and 3' seguences (;nrl~;nr exons 6 and 7); FIXD rontA;nR no naturally occurring introns. Primers (Set 1: Figure 1) srer; T; r to the 5 ' end of the fIX cDNA and 3 ' end of BLG
were designed and constructed. The primers had the following seS!Ie~ces:
Set l-5'~IX (code no. 292343): 5'CAC CAA GCT TCA TCA CCA TCT GCC 3' SCt 1-3'BLG (code ~o. 290646): 5'GGG TGA CTG CAG TCC TGG TCC C 3' ~rrn~A;n~ an introduced .~TIu~dIII site to enable cloning.
These primers amplified the shorter FIXD transcript (named BIX) from the BLG+FIXD mice and this was cloned in plasmid vecto~ rRTTTaq~TPT as pRT-FIX, which was then se~-~nred. The sequence of pRT-FIX showed a 462 nt ;nt~rnAl deletion in the fIX seguences. Thus instead of the 1813 nt size of predicted for FIXD mRNA the BIX
transcripts were 1351 nucleotides (Figure 1).
The sequence of pRT-FIX, determined by the dideoxy method of Sanger, t~f~nT i T-i P-i the precise location of the deletion observed in BIX mRNA. Inspection of the fIX
35 cDNA sequence (Anson et al ., 'rhe EQ~O .Jou~nal 3 (5) 1053-WosS/30000 2 ~ ~ 9 4 38 ` 1~1 . s . -1060 (1984)) and comparison to the 5' and 3' break points deduced from pRT-FIX showed that the deletion was almost certainly due to aberrant splicing. Thus the deletion comprises bp 1085-1547 inclusive (as ' ~d in the Anson paper and in Figure 2 of this sper;f;r~t;~n). The most 5 ' serluPnre is 5 ' GUAAGUGG and the most 3 ' sequence is UUU~:U~:UUUACa~3' (Figure 3). These are very 'good' consensus sequences for the donor (5' ) and acceptor (3' ) sites of an intron. (The 5 ' and 3 ' end8 of an intron must have GU and AG respectively: these are absolute reguirements for spl;r;nr,; the other bases here are also close to the consensus for the donor and acceptor sites.) Note that the presence of donor and acceptor sites does not mean that a gene must be spliced in this way: from the Cpqu~nre one cannot predict whether or not a splice will occur. Indeed in the natural factor IX gene these sites are present in the last exon (exon 8) separated by the same sequences that are in FIXD (Figure 4).
Nevertheles6 these sites are not used in the normal expressing factor IX pre-mRNA in human liver. Thus, for some reason the FIX transcripts produced in the mammary gland use these cryptic splice sites, resulting in the pro~llct;rn of the ;nt~n~lly deleted BIX mRNA. This ;nt~rn~l ly deleted mRNA cannot code for a fllnrt;rn~l fIX
protein since it results in the removal of segment coding for the last 109 amino acids of fIX.
~ ~T~! 2 - Aberrant S~l~rJ"~ Occurs with Other fIX
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The ;dPnt;fication of the aberrant splicing of fI~ cDNA
sequences was made with mice expressing the FIXD
construct (cointegrated with BLG) . Transgenic sheep with fIX cDNA sequences had previously been made, but in these w0 95/30000 ~ g~
2189~3~ ~
, .~, sheep the fIX cDNA sequences were integrated into the f irst exon of the intact Bl,G gene, as a construct called FIXA (as rlPcr~iherl in Example 3 of WO-A-8800239). This construct also appeared to behave rather poorly and produced rather low levels of f IX in the milk . It was, therefore, also of interest to see whether this aberrant splice occurred in the mammary gland with this f IX
construct . Mammary RNA samples f rom sheep carrying another relatively poorly expressing construct, ~FIXA1 (i~n~;f;ed as J FIX A 1 in Section E of Example 4 of WO-A-9005188), were also procured from trAnA~n;c sheep derived f rom a f ounder transgenic prepared as disclosed in WO-A-9005188. A set of PCR primers (Set 2: Figure 5) were ~lP~;r~npt~ which upon RT-PCR -lifirAt;nn of RNA
would distinguish the unspliced fIX sP~1PnrPq from the aherrantly spliced m~NA that was observed for BIX mRNA.
In wild type (non-aberrantly spliced mRNA) these primers would generate a 689 p fragment, whereas in aberrantly spliced mRNA they would generate a 227 bp ~L _ ~.
These primers had the following se~uences:
Set 2-5'fIX ~code no. 795X~: 5' GAG GAG ACA GAA CAT ACA GPG C 3' Set 2-3'fIX (code no. 794X): 5' CAG GTA A~A TAT GAA ATT Cl`C CC 3' and were used against a variety of RNA prepared from tissues expressing fIX. The results are shown in Table 1.

WO95/30000 ~ ~g~ 8 .~ j 6 RNA PCR Splice Comme~t FL _ t Human liver 689 no normal gpl; ~; n~
5 Control m. mammary N/A N/A no fIX expression Control 8 . mammary N/A N/A no f IX expression 8IX (FIXD + BLG) 227 yes rnnf;rTr~Q sequence FIXA: sheep mam 227 yes aberrant splice also FIXA: mouse mam 689 no splice not observed 10 JFIXA1: sheep mam 227 yes aberrant splice also FIXA and JFIXA1 in sheep mammary gland do show the same aberrant splice as BIX, therefore it i8 not strictly construct ~ P~I~- FIXA in mouse does, however, present a rather confusing situation. Only 1/12 mice expressed this construct, but at relatively high levels ~30 ~Lg/ml). The mouse clearly does not carry out this aberrant splice in the mammary gland and hence quite high levels of f IX in milk are seen . But why this happens in

2 0 this one mouse is not understood . Nevertheless it suggests that the absence of the aberrant splice can improve f IX levels in milk .
~;!Y~PT.~;! 3 - Con8trl.~ctios~ of FIX-~3 ' splice This construction is o~ ; nPfl in Figure 6 . A set of PCR
primers (set g) Set 4 5~BIG ~976G) 5~GCT TCT GGG GTC TAC CAG GAA C 3' Set 4 3~ (2212) 5'TAT AAC CCG GGA AAT CCA TCT TTC ATT AP,G T 3~
~t~nn~;n.c additional 5~ sequence ;n~ ;n'J new SmaI site for cloning purposes.

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were used to amplify a segment of FIXD from the 5 ~ BLG
se~uence to a se5~uence just 3' to the stop codon of fIX
but 5 ' to the cryptic acceptor splice site . This segment of DNA thus nnnt~;nR the coding sequence of fIX but lacks the cryptic acceptor site in the 3 ' untr~n~l ~tPfl region.
This segment was fused to BLG se~lpnpc to make a construct very similar to FIXD but lacking 141 bp of 3 ' fl~nk;n~ se~uences of fIX present in FIXD, including the cryptic acceptor site.

l~Y~`~PLE 4 - Expressio~ of FIX-~3 ~ splice To test whether FIX-~3~splice resulted in improved fIX
expression in transgenic animals it was coinjected with BLG into mouse eggs (as per WO-A-9211385~ and a number of transgenic lines es~hl; f~hPd. Expression of the FIX-~3 ' splice transgene was analysed in the mammary gland at the RNA and protein level.
Protei~ an~Lly8i8: Nine lines of transgenic mice have so far been analysed. All of them exhibit ~Ptect~hle levels of fIX in milk. One of them (line 31) showed very high levels (an average of 60 . 9 /lg/ml) with some individuals showing ~100 ILg/ml (Table 2): this is by far the highest level of f IX ever achieved in milk .
F:T,TCP. Analv3i8 of Factor IX Milk Sam~les These milks were from transgenic mice with the modified factor IX cDNA ~acceptor splice site removed). The ELISA
is based on capture by a rabbit polyclonal and detection is by the same polyclonal but modified by biotinylation.
Expression is indicated below:-Wo ss/30000 2 1 8 ~ 1 3 8 1 ~ .3~
TAB~E 2: RNA and Protein Expression in FIX~3 ' Lines Line Copy Nos. RNA Protein BLG/FIXD~3 (ng/llg) ~ ~lg/lll+
5 3 nd + 2.9 ~,2 118/2 +(.04) 4.2 ,3 1215/2 + ( . 02) 9 . 1 8 1414/3 - 0.3 2328/3 - 0.4 2, 1031 6/2 +(.44) 60.9 ~,18)$
34 9/1 - 0.38 (3) 41 6/l - ~0.1 (2) 44 nd + 0.6 (3) ~ estimated by PhosphorImager analysis of S. blots of tail DNA~ these values are apl~roximate ( "nd"
indicates "not done") in some samples the level of FIXD~3 ' mRNA was estimated relative to an in vi tro transcribed f IX
transcript + measured by ELISA; averaged from the number of G
(first generation) or G2 (second generation) samples shown in parf~n~h~cPc $ fIX levels ~Yr~ cl lO0 ~lg/ml in some individuals of this line Furthermore, the protein produced has a very similar mobility to normal plasma derived human f IX on reducing and non-reducing gels (Figure 7) and is biologically active (Table 3). These levels of fIX production would be commercial in sheep.
Purification and Bioloqical Activitv of Hum, n fI~ from T,", ~ L~iC Mouse Milk fIX was purified from pooled mouse milks from line 31 by - ffinity .1~l tor,raphy. MabA7 which binds the Ca+
binding fIX Gla domain was a kind gift from Charles Lutsch. The an~ibody was coupled to cyanogen bromide activated Sepharose. Diluted milk was inrl~h~t~

WO 95/30000 2 1 ~ ~ ~ 3 ~ 5.'~ ., c overnight with antibody-conjugated Sepharose in 50 mM
Tris, 150 mM NaCl pH 7.5 (TBS) + 50 mM CaCl2 at 4C.
Bound protein was eluted isocratically with TBS, 25 mM
EDTA, pH 7.5 fIX coagulation activity was measured by the addition of fIX deficient plasma (Diagnostic Reagents, Oxon, ~) and APTT reagent (Sigma) with the reaction initiated after 5 minutes by addition of Ca+.
Coagulation was measured by ball os~ ti~n with an ST4 Analyser (Diagnostica Stago). Normal human plasma (4 ILg/ml fIX as measured by ELISA) was used as standard.
The results are indicated in Table 3 below:-15 Pooled Milk Elu~te Tot~l flX@ Tot~ flX~iP Rllcovory Concn@ Actlvity+
(~5) l~ls) (pg/ml~ 3/ml) 140 61.6 44% 30.8 30.8 a number of milk sam~les from line FIX~3'31 were pooled measured by ELISA
measured by clotting assay RNA a~ly~ Northern blots of repr~ nt~t~ve RNA
samples from FIx-a3lsplice mice were probed with a fIX-specific probe. The predicted size transcripts (-1680 nt) were ~bselvc:d (Figure 8) and, furt' ~, the steady state mRNA levels correlated with the levels of fIX
detected in milk (eg line 31 had the highest mRNA levels (see Table 2) ) . These FIX-~3 ' splice RWAs were co-run with some BIX ~NAs. Note that they have a higher molecular weight than the BIX mRNA (1351 nt) even though the construct is smaller. The aberrant splice which shortens BIX mRNA has now been cured. This was confirmed Wo 95/3000o ~ 1 8 9 ~ 3 8 by an RT-PCR analy9is of FIX-~3 ' splice RNA which showed that t~e 3 ' segment of the transcript was intact (not shown) .

Claims (11)

16

1. DNA having a sequence encoding a protein having human factor IX activity, wherein the DNA is modified to interfere with the functioning of at least one of the following cryptic splice sites:
(a) a donor site including mRNA nucleotide 1086;
and (b) an acceptor site including mRNA nucleotide 1547;
adopting the mRNA nucleotide numbering of Figure 2 of the drawings.

2. DNA as claimed in claim 1, which encodes wild-type human factor IX.

3. DNA as claimed in claim 1 or 2, which contains at least one of the introns present in genomic DNA encoding factor IX.

4. DNA as claimed in claim 1, 2 or 3, in which the cryptic donor site is engineered out.

5. DNA as claimed in any one of claims 1 to 4, in which the cryptic acceptor site is engineered out.

6. DNA as claimed in claim 5, which is a DNA segment encoding factor IX, the DNA segment being shortened at its 3' end to exclude the acceptor site.

7. DNA as claimed in claim 6, which is cDNA.

8. An expression host comprising DNA as claimed in any one of claims 1 to 7 operably linked to an expression control sequence.

9. An expression host as claimed in claim 8, which is a transgenic non-human animal.

10. An expression host as claimed in claim 9, wherein the animal is a placental mammal and the expression control sequence directs expression in the mammary gland so that factor IX is present in the mammal's milk.

11. An expression host as claimed in claim 9, wherein the expression control sequence comprises the .beta.-lactoglobulin promoter.