AU747522B2

AU747522B2 - Nucleic acid sequence and method for selectively expressing a protein in a target cell or tissue

Info

Publication number: AU747522B2
Application number: AU81999/98A
Authority: AU
Inventors: Ian Frazer; Jian Zhou
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 1997-07-09
Filing date: 1998-07-09
Publication date: 2002-05-16
Anticipated expiration: 2018-07-09
Also published as: AU8199998A

Description

WO 99/02694 PCT/AU98/00530

TITLE

"NUCLEIC ACID SEQUENCE AND METHOD FOR SELECTIVELY EXPRESSING A PROTEIN IN A TARGET CELL OR TISSUE" FIELD OF THE INVENTION THIS INVENTION relates generally to gene therapy. More particularly, the present invention relates to a synthetic nucleic acid sequence and to a method for selectively expressing a protein in a target cell or tissue in which at least one existing codon of a parent nucleic acid sequence encoding the protein has been replaced with a synonymous codon.

The invention also relates to production of virus particles using one or more synthetic nucleic acid sequences and the method according to the invention.

BACKGROUND OF THE INVENTION While gene therapy is of great clinical interest for treatment of gene defects, this therapy has not entered into mainstream clinical practice because selective delivery of genes to target tissues has proven extremely difficult. Currently, viral vectors are used, particularly retroviruses and adenovirus, which are to some extent selective.

However, many vector systems are by their nature unable to produce stable integrants and some also invoke immune responses thereby preventing effective treatment. Alternatively, "naked" DNA is packaged in liposomes or other similar delivery systems. A major WO 99/02694 PCT/AU98/00530 2 problem to be overcome is that such gene delivery systems themselves are not tissue selective, whereas selective targeting of genes to particular tissues would be desirable for many disorders cancer therapy). While use of tissue specific promoters to target gene therapy has been effective in some animal models it has proven less so in man, and selective tissue specific promoters are not available for a wide range of tissues.

The current invention has arisen unexpectedly from recent investigations exploring why papillomavirus (PV) late gene expression is restricted to differentiated keratinocytes. In this regard, it is known that PV late genes L1 and L2 are only expressed in non-dividing differentiated keratinocytes (KCs). Many investigators including the present inventors have been unable to detect significant PV L1 and L2 protein expression when these genes are transduced or transfected into undifferentiated cultured cells, using a range of conventional constitutive viral promoters including retroviral long terminal repeats (LTRs) and the strong constitutive promoters of CMV and PV L1 mRNA can however be efficiently translated in vitro using rabbit reticulocyte cell lysate, suggesting that there are no cellular inhibitors in the lysate interfering with translation of L1. The major difference between the in vitro and in vivo translation systems is that L1 comprises the dominant L1 mRNA in in vitro translation reactions, while it constitutes a minor fraction among the cellular mRNAs in intact cells.

WO 99/02694 PCT/AU98/00530 3 In vivo, PV late proteins are not produced in undifferentiated KC. However, they are expressed in large quantity in highly differentiated KC. The mechanism of this tight control of late gene expression has been poorly understood, and searches by many groups for KC specific PV gene transcriptional

-U

control proteins have been unrewarding.

Blockage to translation of L1 mRNA in vivo has been attributed to sequences within the L1 ORF (Tan et al. 1995, J. Virol. 69 5607-5620; Tan and Schwartz, 1995, J. Virol. 69 2932-2945) By using a Rev and Rev-responsive element of HIV, such inhibition could be overcome (Tan et al. 1995, supra) Accordingly, the inventors examined whether removal of putative "inhibitory sequences" in the L1 ORF would allow production of L1 protein in undifferentiated cells. Deletion mutagenesis of BPV L1 to remove putative inhibitory sequences and expression of resultant deletion mutants in CV-I cells revealed surprisingly that despite expression of L1 mRNA, L1 protein could not be detected.

In view of the foregoing, it has been difficult hitherto to understand how papillomaviruses produce large amounts of L1 protein in the late stage of their life cycle using this apparently "untranslatable" gene.

Surprisingly, however, it has now been discovered that PV L1 protein can be produced at r, substantially enhanced levels in an undifferentiated host cell by replacing existing codons of a native L1 gene with synonymous codons used at relatively high frequency by genes of the undifferentiated host cell 4 compared to the existing codons. It has also been found unexpectedly that there are substantial differences in the relative abundance of particular isoaccepting transfer RNAs (tRNAs) in different cells or tissues and this plays a pivotal role in protein expression from a gene with a given codon usage or composition. This discovery has been reduced to practice in synthetic nucleic acid sequences and generic methods, which utilize codon alteration as a means for targeting expression of a protein to particular cells or tissues or alternatively, to cells in a specific state of differentiation.

OBJECT OF THE INVENTION It is therefore an object of the present invention to provide a synthetic nucleic acid sequence and a method for selectively expressing a protein in a target cell or tissue which sequence and method ameliorate at least some of the disadvantages associated with the prior art.

SUMMARY OF THE INVENTION oo 25 Accordingly, in one aspect of the invention, there is provided a method of constructing a synthetic poiynucleotide from which a protein is selectively expressible in a target cell of a mammal, relative to another cell of the mammal, said method comprising: selecting a first codon of a parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it 5 exhibits a higher translational efficiency in said target cell than in said other cell; and replacing said first codon with said synonymous codon to construct said synthetic polynucleotide.

Preferably, said first codon and said synonymous codon are selected by: comparing translational efficiencies of individual codons in said target cell relative to said other cell; and selecting said first codon and said synonymous codon based on said measurement.

A translational efficiency of a codon may be determined by any suitable technique. In a preferred embodiment, the translational efficiency of a codon is 15 determined by measuring the abundance of an iso-tRNA corresponding to said individual codon in said target cell relative to said other cell.

Preferably, said synonymous codon corresponds

S::

*to an iso-tRNA which is in higher abundance in the target cell relative to said other cell.

Preferably, selecting said first codon and said synonymous codon comprises: measuring abundance of different iso-tRNAs in said *o target cell relative to said other cell; and 25 selecting said first codon and said synonymous codon based on said measurement, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said target cell than in said other cell.

Advantageously, said synonymous codon corresponds to an iso-tRNA that is present in said target cell at a level which is at least 110%, 6 preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of the level that is present in said other cell.

Alternatively, the step of selecting may be characterized in that a synonymous codon according to the invention is selected from the group consisting of a codon used at relatively high frequency by genes of the target cell, a codon used at relatively high frequency by genes of the mammal, a codon used at relatively low frequency by genes of said other cell, a codon used at relatively low frequency by genes of an organism other than said mammal.

In a preferred embodiment, the method further includes the step of selecting the first codon and the synonymous codon such that said protein is expressed from said synthetic polynucleotide in said target cell or tissue at a level which is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of that expressed from said parent polynucleotide in said target cell or tissue.

S. Preferably, the other cell is a precursor cell of the target cell. Alternatively, the other cell may be a cell derived from the target cell.

.In another aspect, the invention provides a synthetic polynucleotide constructed according to any one of the above methods.

Suitably, said synonymous codon(s) are selected from the group consisting of gca (Ala), cuu (Leu) and cua (Leu), when said target cell is a 7differentiated cell, and more preferably when said target cell is a differentiated keratinocyte.

Synonymous codons for higher level expression of a protein in an undifferentiated cell, preferably an undifferentiated keratinocyte, are suitably selected from the group consisting of cga (Arg), cci (Pro) and aag (Asn).

In yet another aspect, the invention resides in a method for selectively expressing a protein in a target cell of a mammal, said method comprising: selecting a first codon of a parent polynucleotide encoding said protein for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a 15 higher translational efficiency in said target cell than in another cell of said mammal; replacing said first codon with said synonymous S. codon to construct a synthetic polynucleotide; and introducing said synthetic polynucleotide into a cell selected from the group consisting of said target cell and a precursor of said target cell, said synthetic polynucleotide being operably linked S* to a regulatory polynucleotide, whereby said protein is selectively expressed in said 25 target cell.

In yet another aspect, the invention provides a method of expressing a protein in a target cell from a first polynucleotide, said method comprising: introducing into a cell selected from the group consisting of said target cell and a precursor of said target cell, a second polynucleotide encoding an iso-tRNA, wherein said second polynucleotide is 8 operably linked to one or more regulatory nucleotide sequences, and wherein said iso-tRNA is normally in relatively low abundance in comparison to other isotRNAs in said target cell and corresponds to a codon of said first polynucleotide, and expressing said second polynucleotide in said target cell, whereby said protein is expressed in said target cell.

In a further aspect, the invention extends to a method of producing a virus particle in a cycling eukaryotic cell, wherein said virus particle comprises a protein necessary for assembly of said virus particle, and wherein said protein is not expressed in said cell from a parent polynucleotide at a level 15 sufficient to permit virus assembly therein, said method comprising: replacing a first codon of said parent polynucleotide with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide, such that said protein is expressible from said synthetic polynucleotide in said cell at a level sufficient to permit virus assembly therein; and introducing into a recipient cell selected from 25 the group consisting of said cycling eukaryotic cell and a precursor of said cycling eukaryotic cell said synthetic polynucleotide operably linked to one or more regulatory nucleotide sequences, whereby said protein is expressed and said virus particle is produced in said cell.

In yet a further aspect of the invention, there is provided a method of producing a virus 9 particle in a cycling cell, wherein said virus particle comprises at least one protein necessary for assembly of said virus particle, wherein said protein is not expressed in said cell from a first polynucleotide at a level sufficient to permit virus assembly therein, and wherein the abundance of an isotRNA specific for a codon of said first polynucleotide limits the rate of production of said protein, said method comprising: introducing into said cell a second polynucleotide encoding said iso-tRNA, and expressing said second polynucleotide in said cell, whereby said virus particle is produced in said cell.

S 15 In still a further aspect, the invention provides a method of constructing a synthetic polynucleotide from which a protein is expressible at a higher level in a first cell of a mammal than in a second cell of the mammal, said method comprising: S 20 selecting a first codon of a parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a higher translational efficiency in said first cell than in said second 25 cell; and t replacing said first codon with said synonymous codon to construct said synthetic polynucleotide.

In yet another aspect of the invention, there is provided a method of constructing a synthetic polynucleotide from which a protein is expressible at a lower level in a first cell of a mammal than in a second cell of the mammal, said method comprising: 9/1 selecting a first codon of a parent polynucleotide for replacement with a synonymous codon which has a lower translational efficiency in said first cell than in said second cell; and replacing said first codon with said synonymous codon to form said synthetic polynucleotide.

In a further aspect, the invention provides a method of expressing a protein in a cell of a mammal, said method comprising: introducing into said cell a vector comprising a synthetic polynucleotide constructed in accordance with the last two-mentioned aspects, wherein said synthetic polynucleotide is operably linked to one or more regulatory nucleotide sequences; and 15 expressing said synthetic polynucleotide in said cell type, whereby said protein is expressed from said synthetic polynucleotide in said cell.

In still yet another aspect, the invention provides a synthetic polynucleotide from which a 20 protein is produced at a higher level in a target cell of a mammal than in another cell of the mammal, *wherein a first codon in a parent polynucleotide encoding said protein has been replaced with a synonymous codon, wherein said synonymous codon has 25 been selected on the basis that exhibits a higher translational efficiency in said target cell than in said other cell, wherein if said synonymous codon is independently selected from the group consisting of gcc, cgc, aac, gac, tgc, cag, ggc, cac, atc, ctg, aag, ccc, ttc, age, acc, tac, gtg, ggg, att, etc, tcc, and gtc, then at least one other codon in said parent polynucleotide has been replaced with another synonymous codon that is not selected from said group.

WO 99/02694 PCT/AU98/00530 BRIEF DESCRITPION OF THE DRAWINGS Figure 1A depicts the nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of BPV1 L1. Amino acids (in single letter code) are presented below the second nucleotide of each codon. Mutations introduced into the genes are indicated above the corresponding nucleotides of the original sequence. Horizontal lines indicate the sites and enzymes used for cloning. This replacement of nucleotides resulted in a nucleic acid sequence encoding BPV-1 L1 polypeptide with an amino acid sequences identical to the wild type, but having synonymous codons that are frequently used by mammalian genes.

Figure 1B shows the nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6) relating to BPV1 L2 ORF. Amino acids (in single letter code) are presented below the second nucleotide of each codon. Mutations introduced into the genes are indicated above the corresponding nucleotides of the original sequence. Horizontal lines indicate the sites and enzymes used for cloning. This replacement of nucleotides resulted in a nucleic acid sequence encoding BPV-1 L2 polypeptide with an amino acid sequences identical to the wild type, but having synonymous codons that are frequently used by mammalian genes.

Figure 1C depicts the nucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID of green fluorescent protein (GFP). Amino acids- (in single letter code) are presented below the SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 second nucleotide of each codon. Mutations introduced into the genes are indicated above the corresponding nucleotides of the original sequence. Horizontal lines indicate the sites and enzymes used for cloning.

This replacement of nucleotides resulted in a nucleic acid sequence encoding GFP polypeptide with an amino acid sequence identical to the native sequence modified for optimal expression in eukaryotic cells, but having synonymous codons that are frequently used by papillomavirus genes.

Figure 2A shows detection of L1 protein expressed from synthetic and wild type BPV1 L1 genes.

Cos-1 cells were transfected with a synthetic L1 expression plasmid pCDNA/HBL1, and a wild type L1 expression plasmid pCDNA/BPVLlwt. The expression of L1 was detected by immunofluorescent staining. Cells were fixed after 36 hrs and incubated with rabbit anti-BPV1 L1 antiserum, followed by FITC-conjugated goat anti-rabbit IgG antibody.

Figure 2B shows detection by Western blot of L1 protein from Cos-1 cells transfected with pCDNA/HBL1 and pCDNA/BPVLlwt.

Figure 2C shows a Northern blot in which L1 mRNA extracted from transfected cells was probed with 3 P-labeled probes produced from wild type L1 sequence.

The amount of mRNA loaded in respective lanes was examined by hybridization of the mRNA sample with a gapdh probe.

Figure 3A shows detection of L2 protein expressed from synthetic and wild type BPV1 L2 genes.

Cos-1 cells were transfected with a synthetic L2 expression plasmid pCDNA/HBL2, and a wild type L2 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 expression plasmid pCDNA/BPVL2wt. The expression of L2 was detected by immunofluorescent staining. Cells were fixed after 36 hrs and incubated with rabbit anti-BPVl L2 antiserum, followed by FITC-conjugated goat anti-rabbit IgG antibody.

Figure 3B shows detection by Western blot of L2 protein from Cos-1 cells transfected with pCDNA/HBL2 and pCDNA/BPVL2wt.

Figure 3C shows a Northern blot in which L2 mRNA extracted from transfected cells was probed with "P-labeled probes produced from wild type L2 sequence.

Figure 4 shows in vitro translation of BPVL1 sequences, wild type BPVL1 (wt) or synthetic L1 (HB) using rabbit reticulocyte lysate or wheat germ extract in the presence of 3 S-methionine. In the top panel, wt L1 or HB L1 plasmid DNA was added to the T7 DNA polymerase-coupled in vitro translation system.

L1 protein was detected by Western blot analysis. In the bottom panel, the translation efficiency of wt L1 or HB L1 sequences in the presence or absence of tRNA was compared. Translation was carried out in rabbit reticulocyte lysate (rabbit) or wheat germ extract (wheat), and samples were collected every two minutes starting from minute 8. Left side of lower panel indicates if 10- 5 M bovine liver or yeast tRNA was supplied.

Figure 5A is a schematic representation of plasmids used to determine L2 expression from BPV cryptic promoter(s). The wild type L1 sequence and SUBSTITUTE SHEET (RULE 26) IA n 1 CnA VV 79/i 4UV t 13 PCTl/AU98/UU53U most of the wild type L2 sequence were deleted from the BPV1 genome by BamHI and HindIII digestion and the remaining BPV1 sequence (in yellow) was cloned into pUC18. Wild type or synthetic humanized L2 sequences (in red) were inserted into the BamHI site of the BPV1 genome. The position of the inserted SV40 ori sequence (in white) is indicated. The plasmid in which modified L2 was used but without SV40 ori sequence was also used as a control. The plasmids were transfected into Cos-1 cells and the expression of L2 protein was determined using BPV1 L2-specific polyclonal antiserum followed by FITC-linked anti rabbit IgG.

Figure 5B shows expression of L2 protein from native papillomavirus promoter. The plasmids shown in Figure 5A were used to transfect Cos-1 cells and the expression of L2 protein was determined using BPV1 L2-specific polyclonal antiserum followed by FITC-linked anti rabbit IgG. A mock transfection in which the cells did not receive plasmid was used as control.

Figure 6 shows expression of GFP in Cos-1 cells transfected with wild-type gfp (wt) or a synthetic gfp gene carrying codons used at relatively high frequency by papillomavirus genes The mRNA extracted from cells transfected with gfp or P gfp was probed with "P-labeled gfp probe and is shown on the right panel, using gapdh as a reference gene.

Figure 7 shows the expression pattern of GFP in vivo from wild-type gfp gene, or a synthetic gfp gene carrying codons used at relatively high frequency by papillomavirus genes. Using a gene gun, SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 mice were shot with PGFP (left panel) and GFP (right panel) expression plasmids encoding GFP protein. A transverse section of the mouse skin section shows where the gfp gene is expressed. Bright-field photographs of the same section where dermis (D) epidermis are highlighted are shown to identify the location of fluorescence in the epidermis. Arrows indicate fluorescent signals.

DETAILED DESCRIPTION The present invention arises from the unexpected discovery that the relative abundance of different isoaccepting transfer RNAs varies in different cells or tissues, or alternatively in cells or tissues in different states of differentiation or in different stages of the cell cycle, and that such differences may be exploited together with codon composition of a gene to regulate and direct expression of a protein to a particular cell or tissue, or alternatively to a cell or tissue in a specific state of differentiation or in a specific stage of the cell cycle. According to the present invention, this selective targeting is effected by replacing at least one existing codon of a parent nucleic acid sequence encoding the protein with a synonymous codon.

Replacement of synonymous codons for existing codons is not new per se. In this regard, we refer to International Application Publication No WO 96/09378 which utilizes such substitution to provide a method of expressing proteins of eukaryotic and viral SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 origin at high levels in in vitro mammalian cell culture systems, the main thrust of the method being the harvesting of such proteins. In distinct contrast, the present invention utilizes substitution of one or more codons in a gene for targeting expression of the gene to particular cells or tissues with the ultimate aim of facilitating gene therapy as described herein.

The term "synonymous codon" as used herein refers to a codon having a different nucleotide sequence to an existing codon but encoding the same amino acid as the existing codon.

By "isoaccepting transfer RNA" is meant one or more transfer RNA molecules that differ in their anticodon structure but are specific for the same amino acid.

Throughout this specification, unless the context requires otherwise, the words "comprise", comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Selection of synonymous codons Determination of relative abundance of different tRNA species in different cells Advantageously, the synonymous codon corresponds to an iso-tRNA (iso-tRNA) which, when compared to an iso-tRNA corresponding to the at least one existing codon, is in higher abundance in the SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 target cell or tissue relative to one or more other cells or tissues of the mammal.

Any method for determining the relative abundance of an iso-tRNA in two or more cells or tissues may be employed. For example, such method may include isolating two or more particular cells or tissues from a mammal, preparing an RNA extract from each cell or tissue which extract includes tRNA, and probing each extract respectively with different nucleic acid sequences each being specific for a particular iso-tRNA to determine the relative abundance of an iso-tRNA between the two or more cells or tissues.

Suitable methods for isolating particular cells or tissues are well known to those of skill in the art. For example, one can take advantage of one or more particular characteristics of a cell or tissue to specifically isolate the cell or tissue from a heterogeneous population. Such characteristics include, but are not limited to, anatomical location of a tissue, cell density, cell size, cell morphology, cellular metabolic activity, cell uptake of ions such as Ca 2 K and H ions, cell uptake of compounds such as stains, markers expressed on the cell surface, cytokine expression, protein fluorescence, and membrane potential. Suitable methods that may be used in this regard include surgical removal of tissue, flow cytometry techniques such as fluorescenceactivated cell sorting (FACS), immunoaffinity separation magnetic bead separation such as Dynabead" separation), density separation metrizamide, Percoll", or Ficoll" gradient SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 centrifugation), and cell-type specific density separation Lymphoprep

M

For example, dividing cells or blast cells may be separated from nondividing cells or resting cells according to cell size by FACS or metrizamide gradient separation.

Any suitable method for isolating total RNA from a cell or tissue may be used. Typical procedures contemplated by the invention are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds) (John Wiley Sons, Inc. 1997), hereby incorporated by reference, at page 4.2.1 through page 4.2.7.

Preferably, techniques which favor isolation of tRNA are employed as, for example, described in Brunngraber, E.F. (1962, Biochem. Biophys. Res.

Commun. 8:1-3) which is hereby incorporated by reference.

The probing of an RNA extract is suitably effected with different oligonucleotide sequences each being specific for a particular iso-tRNA. Of course it will be appreciated that for a given mammal, oligonucleotide sequences would need to be selected which hybridize specifically with particular iso-tRNA sequences expressed by the mammal. Such selection is well within the realm of one of ordinary skill in the art based a known iso-tRNA sequence. For example, in the case of a mouse, exemplary oligonucleotide sequences which may be used include those described in Gauss and Sprinzel (1983, Nucleic Acids Res. 11 hereby incorporated by reference. In this respect, the oligonucleotide sequences may be selected from the group consisting of: SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 -TAAGGACTGTAAGACTT-3' -CGAGCCAGCCAGGAGTC-3' -CTAGATTGGCAGGAATT -3' -TAAGATATATAGATTAT -3' -AAGTCTTAGTAGAGATT-3' -TATTTCTACACAGCATT- 3' -CTAGGACAATAGGAATT-3' -TACTCTCTTCTGGGTTT-3' -TGCCGTGACTCGGATTC -3' -TAGAAATAAGAGGGCTT -3' -TACTTTTATTTGGATTT-3' -TATTAGGGAGAGGATTT -3' -TCACTATGGAGATTTTA-3' -CGCCCAACGTGGGGCTC-3' -TAGTACGGGAAGGATTT-3' -TGTTTATGGGATACAAT-3' -TCAAGAAGAAGGAGCTA-3' -GGGCTCGTCCGGGATTT-3' -ATAAGAAAGGAAGATCG-3' -TGTCTTGAGAAGAGAAG-3' -TGGTAAAAAGAGGATTT-3' -TCAGAGTGTTCATTGGT- 3'

(SEQ

ID NO: 13) ID NO: 14) ID NO:l15) ID NO: 16) ID NO: 17) ID NO: 18) ID NO:l19) ID NO: 20) ID NO:2l) ID NO: 22) ID NO: 23) ID NO: 24) ID NO: 25) I D NO: 26) I D NO: 27) I D NO: 28) I D NO: 29) ID NO: 30) ID NO: 31) ID NO: 32) ID NO: 33) ID NO: 34) for for for for for f or f or for f or for f or for for for for for f or f or f or f or for f or Ala'" Arg

CA

ASnAAC ASpGAC Cys

TGC

Gi u~h GlncA GlywG Hi 5 cAc I leAT LeUCTA LeucT Lys' Lys'~ Me telong Phe

TTC

Procc Proccl SerAGc ThrACA Tyr

TAC

ValGTA Typically, the relative abundance of isotRNA species may be determined by blotting techniques that include a step whereby sample RNA or tRNA extract is immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Northern blotting may be used to identify an RNA sequence that is complementary to a nucleic acid probe. Alternatively, dot blotting SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 and slot blotting can be used to identify complementary DNA/RNA or RNA/RNA nucleic acid sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel, et al (supra) at pages 2.9.1 through 2.9.20.

According to such methods, a sample of tRNA immobilized on a matrix is hybridized under stringent conditions to a complementary nucleotide sequence (such as those mentioned above) which is labeled, for example, radioactively, enzymatically or fluorochromatically.

"Stringency" as used herein, refers to the temperature and ionic- strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between the immobilized nucleotide sequences iso-tRNA) and the labeled oligonucleotide sequence. For a discussion of typical stringent conditions that may be used, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at pages 2.10.1 to 2.10.16, and Sambrook et al in MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), hereby incorporated by reference, at sections 1.101 to 1.104.

While stringent washes are typically carried out at temperatures from about 42 0 C to 68 0

C,

one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions.

Maximum hybridization typically occurs at about 200 to 25' below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 nucleic acid sequences dissociate. Methods for estimating Tm are well known in the art (see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at page 2.10.8).

Maximum hybridization typically occurs at about 100 to 150 below the Tm for a DNA-RNA hybrid.

Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

Methods for detecting labeled nucleotide sequences hybridized to an immobilized nucleotide sequence are well known to practitioners in the art.

Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.

Advantageously, the relative abundance of an iso-tRNA in two or more cells or tissues may be determined by comparing the respective levels of binding of a labeled nucleotide sequence specific for the iso-tRNA to equivalent amounts of immobilized RNA obtained from the two or more cells or tissues.

Similar comparisons are suitably carried out to determine the respective relative abundance of other iso-tRNAs in the two or more cells or tissues. One of ordinary skill in the art will thereby be able to determine a relative tRNA abundance table (see for example TABLE 2) for different cells or tissues. From such comparisons, one or more synonymous codons may be selected such that the or each synonymous codon corresponds to an iso-tRNA which, when compared to an SUBSTITUTE SHEET (RULE 26) iso-tRNA corresponding to an existing codon of the parent nucleic acid sequence, is in higher abundance in the target cell or tissue relative to other cells or tissues of the mammal.

Advantageously, a synonymous codon is selected such that its corresponding iso-tRNA in the target cell or tissue is at a level which is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of that expressed in the or each other cell or tissue of the mammal.

Suitably, synonymous codons for selective expression of a protein in a differentiated cell, preferably a differentiated keratinocyte, are selected 15 from the group consisting of gca (Ala), cuu (Leu) and cua (Leu).

Synonymous codons for selective expression of a protein in an undifferentiated cell, preferably an undifferentiated keratinocyte, are suitably selected from the group consisting of cga (Arg), cci (Pro) and aac (Asn).

Analysis of codon usage Alternatively, synonymous codons may be 25 selected by analyzing the frequency at which codons i. are used by genes expressed in particular cells or tissues, (ii) substantially all cells or tissues of the mammal, or (iii) an organism which may infect particular cells or tissues of the mammal.

Codon frequency tables as well as suitable methods for determining frequency of codon usage in an organism are described, for example, in an article by WO 99/02694 PCT/AU98/00530 Sharp et al (1988, Nucleic Acids Res. 16 8207-8211) which is hereby incorporated by reference.

The relative level of gene expression detectable protein expression vs no detectable protein expression) can provide an indirect measure of the relative abundance of specific iso-tRNAs expressed in different cells or tissues. For example, a virus may be capable of propagating within a first cell or tissue (which may include a cell or tissue at a specific stage of differentiation) but may be substantially incapable of propagating in a second cell or tissue (which may include a cell or tissue at another stage of differentiation). Comparison of the pattern of codon usage by genes of the virus with the pattern of codon usage by genes expressed in the second cell or tissue may thus provide indirectly a set of synonymous codons which correspond to iso-tRNAs expressed at relatively high abundance in the first cell or tissue relative to the second cell or tissue and vice versa. Simultaneously, the above comparison may also provide indirectly a set of synonymous codons which correspond to iso-tRNAs expressed at relatively high abundance in the second cell or tissue relative to the first cell or tissue.

From the foregoing, a synonymous codon according to the invention may correspond to a codon including, but not limited to, a codon used at relatively high frequency by genes, preferably highly expressed genes, of the target cell or tissue, a codon used at relatively high frequency by genes, preferably highly expressed genes, of the or each other cell or tissue, a codon used at relatively SUBSTITUTE SHEET (RULE 26) 111\ anM2 T /AT T11rn //trt in V 23 rL IIA U /aUu3u high frequency by genes, preferably highly expressed genes, of the mammal, a codon used at relatively low frequency by genes of the target cell or tissue, a codon used at relatively low frequency by genes of the or each other cell or tissue, a codon used at relatively low frequency by genes of the mammal, a codon used at relatively high frequency by genes of another organism, and a codon used at relatively low frequency by genes of another organism.

For example, codons used at a relatively high frequency by genes, preferably highly expressed genes, of the mammal may be selected from the group consisting of: cuc (Leu), cuu, (Leu), cug (Leu), uua (Leu), uug (Leu); cgg (Arg), cgc (Arg), aga (Arg), agg (Arg); agu (Ser), age (Ser), ucu (Ser), ucc (Ser), and uca (Ser). Alternatively, such codons may include auu (Ile), auc (Ile); guu (Val), guc (Val), gug (Val); acu (Thr), acc (Thr), aca (Thr); gcu (Ala), gcc (Ala), gca (Ala); cag (Glu); ggc (Gly), gga (Gly), ggg (Gly).

Codons used at a relatively low frequency by genes of the mammal are described, for example, in Sharp et al (1988, supra). Such codons may comprise cua (Leu); cga (Arg), cgu (Arg); ucg (Ser).

Alternatively, such codons may include aua (Ile) gua (Val); acg (Thr); gcg (Ala); caa (Glu); ggu (Gly).

Construction of synthetic nucleic acid sequences The step of replacing synonymous codons for existing codons may be effected by any suitable technique. For example, in vitro mutagenesis methods may be employed which are well known to those of skill in the art. Suitable mutagenesis methods are SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 described for example in the relevant sections of Ausubel, et al. (supra) and of Sambrook, et al., (supra) which are hereby incorporated by reference.

Alternatively, suitable methods for altering DNA are set forth, for example, in U.S. Patent Nos 4,184,917, 4,321,365 and 4,351,901, which are hereby incorporated by reference. Instead of in vitro mutagenesis, the second nucleic acid sequence may be synthesized de novo using readily available machinery. Sequential synthesis of DNA is described, for example, in U.S.

Patent No 4,293,652, which is hereby incorporated by reference. However, it should be noted that the present invention is not dependent on and not directed to any one particular technique for replacing synonymous codons for existing codons.

It is not necessary to replace all the existing codons of the parent nucleic acid sequence with synonymous codons each corresponding to a isotRNA expressed in relatively high abundance in the target cell compared to other cells. Increased expression may be accomplished even with partial replacement. Preferably, the replacing step affects 10%, 15%, 20%, 25%, 30%, more preferably 35%, 60%, 70% or more of the existing codons of the parent nucleic acid sequence.

The parent nucleic acid sequence is preferably a natural gene. By "natural gene" is meant a gene that naturally encodes the protein. However, it is possible that the parent nucleic acid sequence encodes a protein that is not naturally-occurring but has been engineered using recombinant techniques.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 The parent nucleic acid sequence need not be obtained from the mammal but may be obtained from any suitable source such as from a eukaryotic or prokaryotic organism. For example, the parent nucleic acid sequence may be obtained from another mammal or other animal. Alternatively, the parent nucleic acid sequence may be obtained from a pathogenic organism.

In such a case, a natural host of the pathogenic organism is preferably a mammal. For example, the pathogenic organism may be a yeast, bacterium or virus.

For example, suitable proteins which may be used for selective expression in accordance with the invention include, but are not limited to the cystic fibrosis transmembrane conductance regulator (CFTR) protein, and adenosine deaminase (ADA). In the case of CFTR, a parent nucleic acid sequence encoding the CFTR protein which may be utilized to produce the synthetic nucleic acid sequence is described, for example, in Riordan et al (1989, Science 245 1066- 1073), and in the GenBank database under Accession No.

HUMCFTRM, which are hereby incorporated by reference.

The term "nucleic acid sequence" as used herein designates mRNA, RNA, cRNA, cDNA or DNA.

Regulatory nucleotide sequences which may be utilized to regulate expression of the synthetic nucleic acid sequence include, but are not limited to, a promoter, an enhancer, and a transcriptional terminator. Such regulatory sequences are well known to those of skill in the art.

Synthetic nucleic acid sequences according to the invention may be operably linked to one or more SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 regulatory sequences in the form of an expression vector. By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or mammalian or insect virus, into which a synthetic nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including the target cell or tissue or a precursor cell or precursor tissue thereof, or be integratable with the genome of the defined host such that the cloned sequence is reproducible. Thus, by "expression vector" is meant any autonomous element capable of directing the synthesis of a protein. Such expression vectors are well known by practitioners in the art.

The term "precursor cell" as used herein refers to a cell that gives rise to the target cell.

The invention also contemplates synthetic nucleic acid sub-sequences encoding desired portions of the protein. A nucleic acid sub-sequence encodes a domain of the protein having a function associated therewith and preferably encodes at least 10, 20, 100, 150, or 500 contiguous amino acids of the protein.

The step of introducing the synthetic nucleic acid sequence into a target cell will differ depending on the intended use and or species, and may involve non-viral and viral vectors, cationic liposomes, retroviruses and adenoviruses such as, for example, described in Mulligan, (1993 Science 260 926-932) which is hereby incorporated by reference. Such methods may include: SUBSTITUTE SHEET (RULE 26) L/O% a f A9/ 4 DlT'I /A I TOmOQI vvJ 77Iuro y 27 Kr ,IiuL oIuVJU Local application of the synthetic nucleic acid sequence by injection (Wolff et al., 1990, Science 247 1465-1468, which is hereby incorporated by reference), surgical implantation, instillation or any other means. This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the protein encoded by the synthetic nucleic acid sequence so as to increase the effectiveness of that treatment. This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of another factor or factors required for the activity of said protein.

(ii) General systemic delivery by injection of DNA, (Calabretta et al., 1993, Cancer Treat. Rev. 19 169-179, which is hereby incorporated by reference), or RNA, alone or in combination with liposomes (Zhu et al., 1993, Science 261 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is hereby incorporated by reference) or any other mediator of delivery. Improved targeting might be achieved by linking the synthetic nucleic acid sequence to a targeting molecule (the so-called "magic bullet" approach employing for example, an antibody), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the protein produced from said synthetic nucleic acid sequence, or of cells responsive to said protein.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 (iii) Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al., 1987, Mole. Cell Biochem. 7 2745-2752, or of cationic lipids and polyamines: Rose et al., 1991, BioTech. 10 520-525, which articles are hereby incorporated by reference), infection, injection, electroporation (Shigekawa et al., 1988, BioTech. 6 742-751, which is hereby incorporated by reference) or any other way so as to increase the expression of said synthetic nucleic acid sequence in those cells. The modification may be mediated by plasmid, bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993, Science 260 926-932; Miller, 1992, Nature 357 455-460; Salmons et al., 1993, Hum. Gen. Ther. 4 129-141, which articles are hereby incorporated by reference) or other vectors, or other agents of modification such as liposomes (Zhu et al., 1993, Science 261 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is hereby incorporated by reference), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al., 1991, Science 254 1507-1512 and by Dhawan et al., 1991, Science 254 1509-1512, which articles are hereby incorporated by reference. Treated cells may be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.

SUBSTITUTE SHEET (RULE 26)

J.

WO 99/02694 29 PCT/AU98/00530 In yet another aspect, the invention provides a pharmaceutical composition comprising the synthetic nucleic sequences of the invention and a pharmaceutically acceptable carrier.

By "pharmaceutically-acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Any suitable technique may be employed for determining expression of the protein from said synthetic nucleic acid sequence in a particular cell or tissue. For example, expression can be measured using an antibody specific for the protein of interest or portion thereof. Such antibodies and measurement techniques are well known to those skilled in the art.

Applications In one embodiment of the present invention, the target cell is suitably a differentiated cell.

Advantageously, the protein which is desired to be selectively expressed in the differentiated cell is not expressible in a precursor cell thereof (such as an undifferentiated or less differentiated cell of the mammal) from a parent nucleic acid sequence at a level SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 sufficient to effect a particular function associated with said protein. In this embodiment, the step of replacing at least one existing codon with a synonymous codon is characterized in that the synonymous codon corresponds to an iso-tRNA which, when compared to the iso-tRNA corresponding to the at least one existing codon, is in relatively higher abundance in the differentiated cell compared to the precursor cell. Accordingly, a synthetic nucleic acid sequence is produced having altered translational kinetics compared to the parent nucleic acid sequence wherein the protein is expressible in the differentiated cell at a level sufficient to effect a particular function associated with said protein, but wherein the protein is not expressible in the precursor cell at a level sufficient to effect said function.

As used herein, the term "function" refers to a biological, or therapeutic function.

The above embodiment may be utilized advantageously for somatic gene therapy where overexpression of a protein in undifferentiated cells such as stems cells has undesirable consequences including death or differentiation of the stem cells.

In such a case, a suitable protein may include cystic fibrosis transmembrane conductance regulator (CFTR) protein, and adenosine deaminase (ADA).

The differentiated cell may comprise a cell of any lineage including a cell of epithelial, hemopoetic or neural origin. For example, the differentiated cell may be a mature differentiated keratinocyte.

SUBSTITUTE SHEET (RULE 26) \WIO 9/0IOA PrTIr/A TO/RIannn X'n AT r A W V 31 Targeting expression of a protein to progeny of a stem cell but not to the stem cell itself The synthetic nucleic acid sequence produced above may be transfected directly into the differentiated cell for the desired function or alternatively, transfected into the precursor cell.

For example, in the case of ADA deficiency, expression of ADA in stem cells may result in loss of stem phenotype which is undesirable. However, an advantageous therapy may reside in transducing autologous marrow stem cells with a synthetic nucleic acid sequence operably linked to one or more regulatory sequences, wherein existing codons of the wild type ADA gene have been replaced with synonymous codons each corresponding to an iso-tRNA expressed in relatively high abundance in differentiated lymphocytes compared to the marrow stem cells. The transduced stem cells may then be reinfused into the patient. This approach will result in transduced marrow stem cells which are not capable of expressing ADA themselves, but which are able to give rise to a renewable population of differentiated lymphocytes which are capable of expressing ADA at levels sufficient to permit a therapeutic effect. In this regard, a suitable cell source for this purpose may comprise stem cells isolated as CD34 positive cells from a patient's peripheral blood or marrow. For gene delivery, a suitable vector may include a retrovirus or Adeno associated virus.

Alternatively, in the case of inducing cell mediated immunity, dendritic cells are important SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 antigen presenting cells (APC) but have a very limited life span for antigen presentation once activated of between 14 to 21 days. Consequently, dendritic cells provide relatively short-term immune stimulation that may not be optimal. However, in accordance with the present invention, a long-term immune stimulation may be provided by transducing autologous bone marrowderived CD34 positive dendritic cell precursors with a synthetic nucleotide sequence encoding an antigen.

such as the melanoma antigen MART-1, wherein the synthetic sequence is operably linked to one or more regulatory sequences, and wherein existing codons of a wild type nucleotide sequence encoding MART-1 have been replaced with synonymous codons each corresponding to an iso-tRNA expressed in relatively high abundance in dendritic cells compared to the dendritic cell precursors. The transduced dendritic cell precursors may then be reinfused into the patient. This approach will result in transduced dendritic cell precursors which are not capable of expressing MART-1 themselves, but which are able to give rise to a renewable population of dendritic cells which are capable of expressing MART-1 at levels sufficient to permit a lifelong intermittent restimulation of a cytotoxic T lymphocyte (CTL) response to the MART-1 antigen.

Targeting expression of a protein to a stem cell but not to progeny of the stem cell In an alternate embodiment, the target cell may be an undifferentiated cell wherein the protein is not expressible in said undifferentiated cell, from a SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 parent nucleic acid sequence encoding the protein, at a level sufficient to effect a particular function associated with the protein. In such a case, at least one existing codon of the parent nucleic acid sequence is replaced with a synonymous codon corresponding to an iso-tRNA which, when compared to the iso-tRNA corresponding to the at least one existing codon, is in relatively higher abundance- in the undifferentiated cell compared to a differentiated cell. This results in a synthetic nucleic acid sequence having altered translational kinetics compared to said parent nucleic acid sequence wherein the protein is expressible in the undifferentiated cell at a level sufficient to effect a particular function associated with the protein, but wherein the protein is not expressible in differentiated cells derived from the undifferentiated cell at a level sufficient to effect said function.

This alternate embodiment may, by way of example, be used to permit expression of a transcriptional regulatory protein which when expressed in a particular undifferentiated cell or stem cell facilitates differentiation of the stem cell along a particular cell lineage. It will be appreciated that in such a case, the regulatory protein is normally expressed from a gene in which the existing codons correspond to iso-tRNAs which are in relatively low abundance in the stem cell compared to other iso-tRNAs and that therefore the protein is not capable of being expressed at levels sufficient for commitment of the stem cell to differentiate along a particular cell lineage. It will also be apparent that such commitment to differentiate along a SUBSTITUTE SHEET (RULE 26) PCT/AU98/00530 WO 99/02694 particular cell lineage may be utilized to prevent production of a particular lineage of cells such as cancer cells.

Alternatively, the method according to this embodiment may be used to express a transcriptional regulatory protein that is involved in the production of a therapeutic agent or agents. Such a protein may include, for example, NF-kappa-B transcription factor subunit (NF-kappa-B p65) which is involved in the production of interleukin-2 interleukin-3

(IL-

3) and granulocyte and macrophage colony stimulating factor (GMCSF). NF-kappa-B p65 is encoded naturally by a nucleotide sequence comprising a number of existing codons each corresponding to an iso-tRNA expressed in relatively low abundance in stem cells. Accordingly, such sequence may be used as the parent nucleic acid sequence according to this embodiment. A suitable nucleotide sequence encoding this protein is described, for example, in Lyle et al (1994, Gene 138 265-266) and in the EMBL database under Accession No which are hereby incorporated by reference.

A suitable undifferentiated cell which may be utilized in accordance with the present embodiment includes but is not limited to a stem cell, such as a CD34 positive hemopoetic stem cell.

The present embodiment may also be used advantageously for gene therapy where ongoing regulated expression of a transgene is desirable. For example, secure but reversible regulation of fertility is desirable in veterinary practice and in humans.

Such. regulation may be effected by transducing autologous breast ductal epithelial cells with a SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 synthetic nucleic acid encoding a leutinising hormone (LH) antagonist or a leutinising hormone releasing hormone (LHRH) antagonist under the control of one or more regulatory sequences. The synthetic nucleic acid may be produced by replacing existing codons of a parent nucleic acid with synonymous codons corresponding to iso-tRNAs expressed in relatively high abundance in resting breast ductal epithelial cells compared to differentiated cells arising therefrom. Once the transduced cells are implanted back into the patient, expression may be switched off by oral administration of progestagen, forcing the differentiation of the majority of the stem cells and loss of expression of the antagonist. Once pregnancy is established, the suppression would be self sustaining by the naturally produced progestagen. The iso-tRNA composition of resting and oestrogen drived breast epithelial cells may be established by first obtaining resting cells from reduction mammoplasty, and determining the cellular tRNA composition in the presence and absence of oestrogen. The synthetic nucleic acid sequence may be introduced into autologous resting epithelial cells by cell electroporation ex vivo, and the transduced cells may be subsequently transplanted subcutaneously into the patient. Progestagen may be administered as required to reverse regulation of fertility.

Targeting expression of a toxin to a tumor cell but not to any other cells of the mammal Many toxins and drugs are available that can kill tumor cells. However, these toxins and drugs SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 are generally toxic for all dividing cells. This problem may be nevertheless ameliorated by establishing the isoacceptor tRNA composition in a tumor clone, and constructing a synthetic toxin gene ricin gene) or a synthetic anti-proliferation gene the tumor supressor p53) using synonymous codons corresponding to iso-tRNAs expressed at relatively high abundance in the tumor clone compared to normal dividing cells of the mammal. The synthetic gene is then introduced into the patient by suitable means to selectively express the synthetic genes in tumor cells.

Alternatively, a chemotherapy enhancing product gene a drug resistance gene the multi-drug resistance gene) using a codon pattern unlikely to be expressed in the tumor efficiently may be employed.

Targeting gene therapy to control body fat Leptins are proteins known to control satiety. By analogy with animal data, however, if too much leptin is administered to a patient, leptininduced starvation might occur. Advantageously, a synthetic gene encoding leptin may be constructed including synonymous codons corresponding to iso-tRNAs expressed at relatively high levels in activated adipocytes compared to non-activated adipocytes. The synthetic gene may then be introduced into the patient by suitable means such that leptin is only expressed substantially in activated adipocytes as opposed to non-activated adipocytes. As body fat turnover diminishes under the influence of leptin reduced SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 appetite, the metabolic activity of the adipocytes falls and the leptin production decreases correspondingly.

Targeting expression of a protein to a stage of the cell cycle In another embodiment of the invention, the target cell may be a non-cycling cell. In this case, the protein which is- desired to be selectively expressed in the non-cycling cell is expressible in a cycling cell of the mammal from a parent nucleic acid sequence at a level sufficient to effect a particular function associated with the protein. The synonymous codons are selected such that each corresponds to an iso-tRNA which, when compared to the iso-tRNA corresponding to the at least one existing codon, is in higher abundance in the non-cycling cell compared to the cycling cell. Accordingly, a synthetic nucleic acid sequence is produced having altered translational kinetics compared to the parent nucleic acid sequence wherein the protein is expressible in the non-cycling cell at a level sufficient to effect a particular function associated with said protein, but wherein the protein is not expressible in the non-cycling cell to effect said function.

The term "non-cycling cell" as used herein refers to a cell that has withdrawn from the cell cycle and has entered the GO state. In this state, it is well known that transcription of endogenous genes and protein translation are at substantially reduced levels compared to phases of the cell cycle, namely G1, S, G2 and M.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 By "cycling cell" is meant a cell which is in one of the above phases of the cell cycle.

Expressing a protein in a target cell or tissue by in vivo expression of iso-tRNAs in the target cell or tissue In another aspect, the invention extends to a method wherein a protein may be selectively expressed in a target cell by introducing into the cell an auxiliary nucleic acid sequence capable of expressing therein one or more isoaccepting transfer RNAs which are not expressed in relatively high abundance in the cell but which are rate limiting for expression of the protein from a parent nucleic acid sequence to a level sufficient for effecting a function associated with the protein. In this embodiment, introduction of the auxiliary nucleic acid sequence in the cell changes the translational kinetics of the parent nucleic acid sequence such that said protein is expressed at a level sufficient to effect a function associated with the protein.

The step of introducing the auxiliary nucleic acid sequence into the target cell or a tissue comprising a plurality of these cells may be effected by any suitable means. For example, analogous methodologies for introduction of the synthetic nucleic acid sequence referred to above may be employed for delivery of the auxiliary nucleic acid sequence into said cycling cell.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 Assembly of virus particles in cells which do not normally permit assembly of virus particles In yet another aspect, the invention extends to a method for producing a virus particle in a cycling eukaryotic cell. The virus particle will comprise at least one protein necessary for virus assembly, wherein the at least one protein is not expressed in the cell from a parent nucleic acid sequence at a level sufficient to permit virus assembly therein. This method is characterized by replacing at least one existing codon of the parent nucleic acid sequence with a synonymous codon to produce a synthetic nucleic acid sequence having altered translational kinetics compared to the parent nucleic acid sequence such that the at least one protein is expressible from the synthetic nucleic acid sequence in the cell at a level sufficient to permit virus assembly therein. The synthetic nucleic acid sequence so produced is operably linked to one or more regulatory nucleotide sequences and is then introduced into the cell or a precursor cell thereof. The at least one protein is expressed subsequently in the cell in the presence of other viral proteins required for assembly of the virus particle to thereby produce the virus particle.

Advantageously, the synonymous codon corresponds to an iso-tRNA expressed at relatively high level in the cell compared to the iso-tRNAs corresponding to the existing codons.

The cycling cell may be any cell in which the virus is capable of replication. Suitably, the cycling cell is a eukaryotic cell. Preferably, the SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 cycling cell for production of the virus particle is a eukaryotic cell line capable of being grown in vitro such as, for example, CV-1 cells, COS cells, yeast or spodoptera cells.

Suitably, the at least one protein of the virus particle are viral capsid proteins. Preferably, the viral capsid proteins comprise L1 and/or L2 proteins of papillomavirus.

The other viral proteins required for assembly of the virus particle in the cell may be expressed from another nucleic acid sequence(s) which suitably contain the rest of the viral genome. In the case of the at least one protein comprising L1 and/or L2 of papillomavirus, said other nucleic acid sequence(s) preferably comprises the papillomavirus genome without the nucleotide sequences encoding L1 and/or L2.

In yet a further aspect of the invention, there is provided a method for producing a virus particle in a cycling cell, said virus particle comprising at least one protein necessary for assembly of said virus particle, wherein said at least one protein is not expressed in said cell from a parent nucleic acid sequence at a level sufficient to permit virus assembly therein, and wherein at least one existing codon of said parent nucleic acid sequence is rate limiting for the production said at least one protein to said level, said method including the step of introducing into said cell a nucleic acid sequence capable of expressing therein an isoaccepting transfer RNA specific for said at least one codon.

SUBSTITUTE SHEET (RULE 26) lir\ ftI-n'i* A lr~f'T'/r l T r'hoIz4 /LAzA W 4 77/u.oUY 41 I/AUYOuu41 In yet a further aspect, the invention resides in virus particles resulting from the above methods.

The invention further contemplates cells or tissues containing therein the synthetic nucleic acid sequences of the invention, or alternatively, cells or tissues produced from the methods of the invention.

The invention is further described with reference to the following non-limiting examples.

EXAMPLE 1 Expression of synthetic L1 and L2 protein in undifferentiated cells.

Materials and Methods Codon replacements in the bovine PV (BPV) L1 and L2 genes The DNA and amino acid sequences of the wild-type L1 (SEQ ID NOS:l,2)and L2 genes (SEQ ID NOS:5,6) are shown respectively in Figures 1A and 1B.

To determine whether the presence of rare codons in wild-type L1 (SEQ ID NO:1) and L2 (SEQ ID NO:5) genes (Table 1) inhibited translation, we synthesized the L1 (SEQ ID NO:3) and L2 (SEQ ID NO:7) genes by using synonymous substitutions as shown. To construct the synthetic sequences, we synthesized 11 pairs of oligonucleotides for L1 and 10 pairs of oligonucleotides for L2. Each pair of oligonucleotides has restriction sites incorporated to facilitate subsequent cloning (Figures 1A and 1B).

The degenerate oligonucleotides were used to amplify SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 L1 and L2 sequences by PCR using a plasmid with BPV1 genome as the template. The amplified fragments were cut with appropriate enzymes and sequentially ligated to pUC18 vector, producing pUCHBL1 and pUCHBL2. The synthetic L1 (SEQ ID NO:3) and L2 (SEQ ID NO:7) sequences were sequenced and found to be error-free, and then sub-cloned into the mammalian expression vector pCDNA3 containing SV40 ori (Invitrogen), giving expression plasmids pCDNA/HBL1 and pCDNA/HBL2. To compare expression of L1 and L2 with that of the original sequences, the wild type L1 (SEQ ID NO:1) and L2 (SEQ ID NO:5) genes were cloned into the pCDNA3 vector, resulting in pCDNA/BPVLlwt and pCDNA/BPVL2wt.

Immunofluorescence and Western blot staining For immunoblotting assays, Cos-1 cells in 6-well plates were transfected with 2 Mg L1 or L2 expression plasmids using lipofectamine (Gibco). 36 hrs after transfection, cells were washed with 0.15M phosphate buffered 0.9% NaCi (PBS) and lysed in SDS loading buffer. The cellular proteins were separated by 10% SDS PAGE and blotted onto nitrocellulose membrane. The L1 or L2 proteins were identified by electrochemiluminescence (Amersham, UK), using BPV1 L1 (DAKO) or L2-specific (17) antisera. For immunofluorescent staining, Cos-1 cells were grown on 8-chamber slides, transfected with plasmids, and fixed and permeabilised with 85% ethanol 36hr after transfection. The slides were blocked with 5% milk-PBS and probed with L1 or L2-specific antisera, followed by FITC-conjugated anti-rabbit IgG (Sigma). For GFP or SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 PGFP plasmid transfected cells, the cell were fixed with 4% buffered formaldehyde and viewed by epifluorescence microscopy.

Northern blotting Cos cells transfected with various plasmids were used to extract cytoplasmic or total RNA using the QIAGEN RNeasy mini kit according to the supplier's handbook. Briefly, for cytoplasmic RNA purification, buffer RLN (50 mM Tris, pH 8.0, 140 mM NaCl, 1.5 mM MgC1 2 and 0.5% NP40) was directly added to monolayer cells and cells were lysed in 4 OC for 5 min.

After the nuclei were removed by centrifugation, cytoplasmic RNAs were purified by column. For total RNA extraction, the monolayer cells were lysed using buffer RLT supplied by the kit and RNA was purified by spin column. The purified RNAs were separated by agarose gel in the presence of formaldehyde. The RNAs were then blotted onto nylon membrane and probed with 1:1 mixed 5'-end labelled L1 wt and HBL1 fragments; 1:1 mixed 5'-end labelled L2 wt and HBL2 fragments; 1:1 mixed 5'end labelled GFP and PGFP fragments or randomly labelled PAGDH fragment. The blots were washed extensively at 65 OC and exposed to X-ray films for three days.

Results To test the hypothesis that the codon composition of the genes encoding the L1 and L2 capsid proteins of papillomavirus (PV) contributes to their preferential expression in differentiated epithelial SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 cells, we produced synthetic BPV1 L1 (SEQ ID NO:3) and L2 (SEQ ID NO:7) genes, substituting codons preferentially used in mammalian genes for the codons frequently present in the wild type BPV1 L1 and L2 sequences which are rare in eukaryotic genes (Figures 1A, 1B).

For the L1 gene, a total of 202 base substitutions were made in 196 codons, without changing the encoded amino acid sequence (Figure 1A).

This synthetic "humanized" BPV L1 gene (SEQ ID NO:3) was designated HBL1. In a similarly modified BPV1 L2 gene (SEQ ID NO:7) designated HBL2, 303 bases were changed to substitute 290 less frequently used codons with the corresponding preferentially used codons.

Using the synthetic HBL1 (SEQ ID NO:3) and HBL2 (SEQ ID NO:7) genes, we constructed two eukaryotic expression plasmids based on pCDNA3, and designated pCDNA/HBL1 and pCDNA/HBL2. Similar expression plasmids, constructed with the wild type BPV1 L1 (SEQ ID NO:1) and BPV1 L2 (SEQ ID NO:5) genes, were designated pCDNA/BPVLlwt and pCDNA/BPVL2wt, respectively. In each of these plasmids the SV40 ori allowed replication in Cos-1 cells, and the L1 or L2 gene was driven by a strong constitutive CMV promoter.

To compare the expression of the synthetic humanized and the wild type BPV1 L1 or BPV1 L2 genes, we separately transfected Cos-1 cells with each of the L1 and L2 plasmids described above. Transfected cells were analyzed for expression of L1 (SEQ ID NO:2,4) or L2 (SEQ ID NO:6,8) protein by immunofluorescence 36 hr after transfection (Figures 2A and 3A) Cells transfected with the pCDNA3 expression plasmid SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 containing the synthetic humanized L1 (SEQ ID NO:3) or L2 (SEQ ID NO:7) genes were observed to produce large amounts of the corresponding protein, while cells transfected with expression plasmids with the wild type L1 (SEQ ID NO:1) or L2 (SEQ ID NO:5) sequences produced no detectable L1 or L2 protein (Figures 2A and 3A, see nuclear staining of L1 and L2 proteins).

To compare more accurately the expression of the different L1 and L2 constructs, L1 and L2 protein expression was assessed by immunoblot in Cos-1 cells transfected with the wild type or synthetic humanized BPV1 L1 or L2 pCDNA3 expression constructs (Figures 2B and 3B) Large amounts of immunoreactive L1 and L2 proteins were expressed from the synthetic humanized L1 (SEQ ID NO:3) and L2 (SEQ ID NO:7) sequences, but no L1 or L2 protein was expressed from the wild type L1 and L2 sequences (SEQ ID To establish whether the alterations to the primary sequence of the L1 and L2 mRNA which resulted from the codon alterations also affected steady state expression of the corresponding message, mRNA was prepared from Cos-1 cells transfected with the various capsid protein gene constructs. Using GAPDH as an internal standard it was established by Northern blot that two to three times more modified than wild type L1 mRNA, and similar levels of wild type and modified L2 mRNA were present in the cytoplasm of transfected cells (Figures 2C and 3C). The amount of L1 or L2 protein expressed per arbitrary unit of L1 or L2 mRNA was at least 100 fold higher for the humanized gene constructs than for the natural gene constructs.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 EXAMPLE 2 Papillomavirus late protein translation in vitro Materials and Methods In vitro translation assay One microgram of each plasmid was incubated with 20 ACi 3 5 S-methionine (Amersham) and iL T7 coupled rabbit reticulocyte or wheat germ lysates (Promega). Translation was performed at 30 °C and stopped by adding SDS loading buffer. The L1 proteins were separated by 10% SDS PAGE and examined by autoradiography.

Production of aminoacyl-tRNA x 10- 4 M tRNA (Boehringer) was added to a 20 AL reaction containing 10 mM Tris-acetate, pH.7.8, 44 mM KC1, 12 mM MgCl 2 9 mM -mercaptoethanol, 38 mM ATP, 0.25 mM GTP and 7 AL rabbit reticulocyte extract. The reaction was carried out at 25 OC for min, and 30 AL H 2 0 was added to the reaction to dilute the tRNAs to 1 x 10" 4 M. The aminoacyl-tRNAs were then aliquoted and stored at -70 oC.

Results As the major limitation to expression of the wild type BPV L1 and L2 genes appeared to be translational in our system we wished to test whether this limitation reflected a limited availability of the appropriate tRNA species for gene translation. As transient expression of the synthetic genes within SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 intact cells may be regulated by many factors, we tested our hypothesis in a cell free system using rabbit reticulocyte lysate (RRL) or wheat germ lysate to examine gene translation. Similar amounts of plasmids expressing the wild type or synthetic humanized BPV1 L1 gene were added to a T7-DNA polymerase coupled RRL transcription/translation system in the presence of "S-methionine. After minutes, translated proteins were separated by SDS PAGE and visualized by autoradiography. Efficient translation of the modified L1 gene was observed (Figure 4, top panel, lane while translation of the wild type BPV1 L1 sequence resulted in a weak kDa L1 band (Figure 4, upper panel, lane We reasoned that although the wild type sequence was not optimized for translation in RRL, some translation would occur as there would be no cellular mRNA species competing for the 'rare' codons present in the wild type L1 sequence. The above data suggest that the observed difference in efficiency of translation of the wild type and synthetic humanized L1 genes is a consequence of limited availability of the tRNAs required for translation of the rare codons present in the wild type gene. We therefore expected that addition of excess tRNA to the in vitro translation system would overcome the inhibition of translation of the wild type L1 gene. To address this question, 10-5 M aminoacyl-tRNAs from yeast were added into the RRL translation system, and L1 protein synthesis was assessed. Introduction of exogenous tRNAs resulted in a dramatic improvement in translation of the wild type L1 sequence, which now gave a yield of L1 protein SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 comparable to that observed with the synthetic humanized L1 sequence (SEQ ID NO:3) (Figure 4, top panel). Enhancement of translation of the wild type L1 gene (SEQ ID NO:1) by aminoacyl-tRNA was dosedependent, with an optimum efficiency at 10 5 M tRNA.

As addition of exogenous tRNA improved the yield of L1 protein translated from the wild type L1 gene sequence (SEQ ID NO:1), we assessed the speed of translation of wild type and humanized L1 mRNA. Samples were collected from the translation mixture every 2 minutes, starting at the 8th minute. Translation of L1 (SEQ ID NO:2,4) from the wild type sequence (SEQ ID NO:1) was much slower than from the humanized L1 sequence (SEQ ID NO:3) (Figure 4 bottom panel), and the retardation of translation could be completely overcome by adding exogenous tRNA from commercially available. yeast tRNA. Yeast tRNA was chosen in the above analysis because the codon usage in yeast is similar to that of papillomavirus (Table Addition of exogenous tRNA did not significantly improve the translation of the humanized L1 gene (SEQ ID NO:3), indicating that this sequence was optimized with regard to codon usage for the rabbit reticulocyte translation machinery (Figure 4, bottom panel) In separate experiments we established that wt L1 translation could also be enhanced by liver tRNA (Figure and by tRNAs extracted from bovine skin epidermis, which presumably constitutes a mixture of tRNAs from differentiated and undifferentiated cells (data not shown).

SUBSTITUTE SHEET (RULE 26) IWOr n/026n4 DrTI /A In/n Inin VTv \771iViuh 49 1 /R~II 7oI/Jv9 EXAMPLE 3 Translation of wild type L1 is efficient in wheat germ extract.

To further test our hypothesis that tRNA availability is a determinant of expression of the wild type BPV1 L1 gene (SEQ ID NO:1), we examined the translation of L1 in a cell type in which a quite different set of tRNAs would be available. In a wheat germ translation system, wild type L1 mRNA was translated as efficiently as humanized L1 mRNA, and addition of exogenous aminoacyl-tRNAs did not improve the translation efficiency of either wild type or humanized sequences (Figure 4 bottom panel). This indicated that in wheat germ there are sufficient of the tRNAs which are limiting for translation of wild type L1 sequence in RRL to allow efficient L1 translation.

EXAMPLE 4 Modified late genes can be expressed in undifferentiated cells from papillomavirus promoter(s) While our data presented above indicates that translation is limiting for the production of BPV1 capsid proteins in our test system, these experiments were conducted in systems which are not truly representative of the viral late gene transcription from the BPV genome, in part because the genes were driven by a strong CMV promoter. We therefore wished to establish whether synthetic SUBSTITUTE SHEET (RULE 26) IL/ DPTIr/ A I n ,nn-n V J 4y/U4uy* 5 0 '-IJ7OIuuJ.0J humanized BPV capsid protein mRNA would be translated more efficiently than the wild type mRNA, if transcribed from the natural BPV1 promoter. This would establish whether translation was indeed one of the limiting factors for expression of BPV1 late genes driven from the natural cryptic late gene promoter in an undifferentiated cell. The BPV genome was cleaved at nt 4450 and 6958 with BamHI/HindIII and the original L1 (nt 4186-5595) and L2 (5068-7095) ORFs were removed. The synthetic humanized L2 gene (SEQ ID NO:7), together with an SV40 ori sequence to allow plasmid replication in eukaryotic cells, were inserted into the BPV genome lacking L1/L2 ORF sequences. This plasmid (Figure 5A) was designated pCICR1. A similar plasmid was constructed with wild type (SEQ ID rather than synthetic humanized L2 and designated pCICR2. Cos-1 cells were transfected with these plasmids and L2 protein expression examined by immunofluorescence of transfected cells. Synthetic humanized L2 (SEQ ID NO:7), driven by the natural BPV- 1 promoter, was efficiently expressed, whereas the wild type L2 sequence (SEQ ID NO:5), driven from a similar construct, produced no immunoreactive L2 protein (SEQ ID NO:6,8) (Figure 5B). As undifferentiated cells supported the expression of the humanized L2 gene (SEQ ID NO:7) but not the wild type L2 (SEQ ID NO:5) expressed from the cryptic late BPV promoter, the results confirmed our earlier observations from experiments using the CMV promoter.

However, the plasmids tested here contained SV40 ori, designed to replicate the DNA in Cos cells. The increased copy number of the BPV1 L2 plasmids or the SUBSTITUTE SHEET (RULE 26) \i a/0AKAA IDPTI/A inQI/naC 51 1 I V JOIVU .V transcriptional enhancing activity of the SV40 ori might explain in part the increased efficiency of expression of L2 in this experimental system when compared with infected skin. However, the marked difference in expression between the natural and humanized genes seen with a CMV promoter construct is still observed with the natural promoter.

EXAMPLE Substitution of papillomavirus-preferred codons prevents translation but not transcription of a nonpapillomavirus gene in undifferentiated cells.

Materials and Methods Codon replacement in gfp gene To construct a modified gfp gene (SEQ ID NO:11) using papillomavirus preferred codons (PGFP), 6 pairs of oligonucleotides were synthesized. Each pair of oligonucleotides has restriction sites incorporated and was used to amplify gfp using a humanized gfp gene (SEQ ID NO:9) (GIBCO) as template. The PCR fragments were ligated into the pUC18 vector to produce pUCPGFP.

The PGFP gene was sequenced, and cloned into BamHI site of the same mammalian expression vector, pCDNA3, under the CMV promoter. The DNA and deduced amino acid sequences of the humanized GFP gene are shown in Figures 1C. Mutations introduced into the wild type gfp gene (SEQ ID NO:9) to produce the Pgfp gene (SEQ ID NO:11) are indicated above the corresponding nucleotides of the wild-type sequence.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 Results To further confirm that codon usage can alter gene expression in mammalian cells, we made a further variant on a synthetic gfp gene modified for optimal expression in eukaryotic cells (Zolotukhin, et al., 1996. J. Virol. 70:4646-4654). In our variant, codons optimized for expression in eukaryotic cells were substituted by those preferentially used in papillomavirus late genes. Of 240 codons in the humanized gfp gene (SEQ ID NO:9), which expresses high levels of fluorescent protein in cultured cells, 156 were changed to the corresponding papillomavirus late gene-preferred codons to produce a new gfp gene (SEQ ID NO:11) designated Pgfp. Expression of Pgfp (SEQ ID NO:11) in undifferentiated cells was compared with that of humanized gfp (SEQ ID NO:9). Cos-1 cells transfected with the humanized gfp (SEQ ID NO:9) produced a bright fluorescent signal after 24 hrs, while cells transfected with Pgfp (SEQ ID NO:11) produced only a faint fluorescent signal (Figure 6A).

To confirm that this difference reflected differing translational efficacy, gfp specific mRNA was tested in both transfections and found not to be significantly different (Figure Thus, codon usage and corresponding tRNA availability apparently determines the observed restriction of expression of PV late genes, and modification of codon usage in other genes similarly prevents their expression in undifferentiated cells.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 PGFP with papillomavirus-preferred codons is efficiently expressed in vivo in differentiated mouse keratinocytes.

Materials and Methods Delivery of plasmid DNA into mouse skin by gene gun Fifty microgram of DNA was coated onto ig gold micro-carriers by calcium precipitation, following the manufacturer's instructions (Bio-Rad).

C57/bl mouse skin was bombarded with gold particles coated with DNA plasmid at a pressure of 600 psi.

Serial sections were taken from the skin and examined for distribution of the particles, confirming that a pressure of 600 psi could deliver particles throughout the epidermis.

Results Mice were shot with gold beads carrying PGFP DNA plasmid and, 24 hrs later, skin samples were cut from the site of DNA delivery and examined for expression of GFP protein (SEQ ID NO:10,12).

Fluorescence was detected mostly in upper keratinocyte layers, representing the differentiated epithelium, and was not seen in undifferentiated basal cells. In contrast, skin sections shot with the humanized GFP plasmid showed fluorescence in cells randomly distributed throughout the whole epidermis (Figure 7).

Although GFP-positive cells were rare in both PGFP- (SEQ ID NO:11) and GFP-inoculated (SEQ ID NO:9) mouse SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 skin, fluorescence was observed only in differentiated strata in the PGFP sample (SEQ ID NO:11), whereas fluorescence was observed throughout the epidermis in GFP-inoculated (SEQ ID NO:9) mouse skin. This result confirmed that the use of papillomavirus-preferred codons resulted in the protein being expressed in an epithelial differentiation-dependent manner.

EXAMPLE 7 Microinjection of yeast tRNA and wild type L1 gene into cultured cells To test if yeast tRNA could facilitate expression of wild type BPV-1 L1 (SEQ ID NO:1) (as yeast uses a similar set of codons to those observed in papillomavirus for its own genes), 2 pL of mixtures containing tRNA (2 mg/mL) (purified yeast tRNA (Boehringer Mannheim) or bovine liver tRNA control) and BPV L1 DNA (2 Ag/mL) can be injected into CV-1 cells (Lu and Campisi, 1992, Proc. Natl. Acad.

Sci. U. S. A. 89 3889-3893). The injected cells can then be cultured for 48 hrs at 37 OC and examined for expression of L1 gene by standard immunofluoresence methods using BPV Li-specific antibody and quantified by FACS analysis (Qi et al 1996, Virology 216 35-45).

EXAMPLE 8 Establishment of a cell line which can continuously produce HPV virus particles To produce infectious PV, various methods have been tried including the epithelial raft culture SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 system (Dollard et al 1992, Genes Dev 6 1131-1142), and cell lines containing BPV-1 episomal DNA, and infected by BPV-1 L1/L2 recombinant vaccinia (Zhou et al 1993, J. Gen. Virol. 74 763-768) or transfected by SFV RNA (Roden et al 1996, J. Virol. 70 5875-5883).

The yield of particles is in each case low. In a reduction to practice of our discovery, synthetic BPV L1 (SEQ ID NO:3) and L2 genes (SEQ ID NO:7) (as described in Example 1) can be used to produce infectious BPV in a cell line containing BPV-1 episomal DNA. Fibroblast cell lines (CON/BPV) containing BPV-1 episomal DNA (Zhou et al 1993, J.

Gen. Virol. 74 763-768) can be used for transfection of the synthetic BPV-1 L1 (SEQ ID NO:3) and L2 genes (SEQ ID NO:7) under control of CMV promoter. BPV particles may then be purified from the cell lysate and the purified particles examined for the presence of BPV-1 genome. Standard methods such as transfection with lipofectamine (BRL) and G418 selection of transfected cells can be utilized to generate suitable transfectants expressing humanized L1 (SEQ ID NO:3) and L2 (SEQ ID NO:7) in the background of BPV-1 episomal DNA. Examination of L1 and L2 protein expression can be performed using rabbit anti-BPV L1 or rabbit anti-BPV L2 polyclonal antibodies. BPV particles can then be purified using our published methods (Zhou et al 1995, Virology 214 167-176) and can be characterized by electron microscopy and DNA blotting. The infectivity of BPV particles isolated from the cultured cells may be tested in focus formation assays using C127 fibroblasts.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 Method for extracting and measuring tRNA from tissues Tissue(100g) is homogenized in a Waring Blender with 150 mL of phenol (Mallinckrodt, Analytical Reagent, 88%) saturated with water (15:3) and 150 mL of 1.0 M NaCi, 0.005 M EDTA in 0.1 M Trischloride buffer, pH 7.5. The homogenate was spun for ten minutes at top speed in the International clinical centrifuge and the upper layer was carefully decanted off. To this aqueous layer, three volumes of ethanol were added. The resultant precipitate was spun down at top speed in the International clinical centrifuge and resuspended in 250 mL of 0.1 M Tris/chloride buffer, pH 7.5. This solution was added (flow rate of 15-20 drops per minute) to a column (2 x cm) of 2 g of DEAE-cellulose previously equilibrated with cold 0.1 M Tris-chloride buffer pH The column was then washed with 1 L of Trischloride buffer, pH 7.5 and the RNA eluted with 1.0 M NaCl in 0.1 M Tris-chloride buffer, pH 7.5. The first mL of NaCl solution were discarded as "hold-up." Sufficient salt solution (60-80 mL) was then collected until the optical density of the effluent was less than three at 260 nm. This solution was extracted twice with an equal volume of phenol saturated with water and twice with ether. To the aqueous solution containing the RNA, three volumes of 95% ethanol were added and the solution wag allowed to stand overnight in the cold. The precipitate was spun down and washed first with 80% and then twice with 95% ethanol and SUBSTITUTE SHEET (RULE 26) WO 99/02694 57 PCT/AU98/00530 dried in a vacuum. Approximately 60 mg of soluble RNA were obtained from a 100-g lot of rat liver.

Quantitating tRNAs The following nylon membranes are used: Biodine A and B (PALL). For the preparation of dot blots, the tRNA samples (from 1 pg to 5 ng) are denatured at 60 oC for 15 min in 1-5 AL of formaldehyde. 10x SSC (SSC is NaCl 0.3 M, tri-sodium citrate 0.03 The samples are spotted in 1 AL aliquots onto the membranes that have been soaked for min in deionized water and slightly dried between two sheets of 3MM Whatman paper prior to the application of the samples. The tRNAs are fixed covalently (in the membranes by ultravioletirradiation (10 mm using an ultraviolet lamp at 254 nm and 100 W strength at a distance of 20 cm) and the membranes are baked for 2-3 h at 80 OC.

A 5' end labelled synthetic deoxyribooligonucleotide complementary to the A54-A73 sequence of the tRNA is used as a probe for the hybridization experiments. Labelling of the oligonucleotide is performed by direct phosphorylation of the 5' OH' ended probe.

For hybridisation experiments, the UVirradiated membranes are first preincubated for 5 h at C in 50% deionized formamide, 5 x SSC, 1% SDS, 0.04% Ficoll 0.04% polyvinylpyrrolidone and 250 jtL/mL of sonicated salmon sperm DNA using 5 mL of buffer for 100 cm 2 of membrane. Hybridization is finally performed overnight at 50 OC in the above solution mL/100 cm 2 where the labeled probe has been SUBSTITUTE SHEET (RULE 26) tl WO 99/02694 PCT/AU98/00530 added. After hybridization, the membranes are washed twice in 2 x SSC, 0.1% SDS for 5 min at room temperature, twice in 2 x SSC, 1% SDS for 30 mm at oC and finally in 0.1 x SSC. 0.1% SDS for 30 min at room temperature. To detect the hybridized probes the membranes are exposed for 16 h to Fuji XR film at 70 C with an intensifying screen.

Sequence of tRNA probes follows: Ala" ArgCGA AsnAA AspGAc CsyTG c Glu GlnAA GlyGGA: Hiscc: Il eATC: LeuCTA: Leu Leuc": LysA

A

LysAAG: Lys Metel"on PheTT procc Procc SerAG ThrACA: TyrTAc: The sequences of the 5' -TAAGGACTGTAAGACTT 5'-CGAGCCAGCCAGGAGTC 5' CTAGATTGGCAGGAATT 5' -TAAGATATATAGATTAT 5' -AAGTCTTAGTAGAGATT 5' -TATTTCTACACAGCATT 5' -CTAGGACAATAGGAATT 5' -TACTCTCTTCTGGGTTT 5'-TGCCGTGACTCGGATTC 5' -TAGAAATAAGAGGGCTT 5' -TACTTTTATTTGGATTT 5' -TATTAGGGAGAGGATTT 5' -TCACTATGGAGATTTTA 5' -CGCCCAACGTGGGGCTC 5' -TAGTACGGGAAGGATTT 5' -TGTTTATGGGATACAAT 5' -TCAAGAAGAAGGAGCTA 5'-GGGCTCGTCCGGGATTT 5' -ATAAGAAAGGAAGATCG 5' -TGTCTTGAGAAGAGAAG 5' -TGGTAAAAAGAGGATTT (SEQ I (SEQ I (SEQ I (SEQ I (SEQ I (SEQ I (SEQ I

(SEQ

D

:D

D

ID

II

ID

II

I]

I

I:

I

NO: 13) NO: 14) NO: NO:16) NO: 17) NO: 18) NO: 19) NO:21) NO:22) i NO:23) NO:24) SNO:26) NO:27) D NO:28) D NO:29) D D NO: 31) D NO:32) D NO:33) tRNA probes are as SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 ValGTA: 5'-TCAGAGTGTTCATTGGT (SEQ ID NO:34) EXAMPLE Comparison of the relative abundance of tRNA species in undifferentiated and differentiated keratinocytes Materials and Methods Isolation of epidermal cells 2-day old mice were killed and their skins removed. The skins were digested with 0.25% trypsin PBS at 4 OC overnight. The epidermis was separated from the dermis using forceps and minced with scissors in 10% FCS DMEM medium. The cell suspension was first filtered through a 1 mm and then a 0.2 mm nylon net.

The cell suspension was then pelleted and washed twice with PBS.

Density gradient centrifugation The keratinocytes were resuspended in Percoll and separated by centrifugation through a discontinuous Percoll gradient (1.085, 1.075 and 1.050 g/mL) at 1200 x g at room temperature for 25 min. The cells were then washed with PBS and used to extract tRNA.

tRNA purification The cells were lysed in 5 mL of lysis buffer (0.2 M NaOH, 1% SDS) for 10 min at room temperature. The lysate was neutralized with 5 mL of 3.0 M potassium acetate (pH After centrifugation, the supernatant was diluted with 3 SUBSTITUTE SHEET (RULE 26) volumes of 100 mM Tris (pH 7.5) and added to a DEAE column equilibrated with 100 mM Tris (pH An equal volume of isopropanol was added to the aqueous solution containing tRNA, and the solution was allowed to stand overnight at 4 oC. The tRNA was spun down and washed with 75% ethanol, then dissolved in RNasefree water.

tRNA blotting 10 ng of each tRNA sample in 1 pL was denatured in 600C for 15 min in 4 iL formaldehyde and A.L 20 x SSC. The samples were spotted in 1 pL aliquots onto charged nylon membrane (Amersham), and the tRNAs were fixed with UV and probed with 32 p_ 15 oligonucleotides.

Results 9 S. Comparison of the abundance of the tRNA species in undifferentiated and differentiated keratinocytes showed that the levels of some tRNA 20 populations changed dramatically. For example, the levels of tRNAs specific for AlaGCA, LeucTT, LeuCTA were 9 increased in differentiated cells while tRNAs for CGA CCI AAC Arg

A

Pro c AsnA were more abundant in 9 undifferentiated keratinocytes (see Table 2).

S* 9 GENERAL DISCUSSION In the present specification the inventors have confirmed that one determinant of the efficiency of translation of a gene in mammalian cells is its codon composition. This observation has commonly been PCT/AU98/00530 WO 99/02694 made when genes from prokaryotic organisms have been expressed in eukaryotic cells (Smith, D. 1996, Biotechnol. Prog. 12:417-422). The present inventors have also presented evidence that mRNA encoding the capsid genes of papillomavirus are not effectively translated in cultured eukaryotic cells, apparently because tRNA availability is rate limiting for translation, and that the block to PV late gene translation in eukaryotic cells in culture can be overcome by altering the codon usage of the late genes to match the consensus for mammalian genes, or alternatively by providing exogenous tRNAs.

Alterations to mRNA secondary structure or protein binding (Sokolowski, et al., 1998, J. Virol. 72:1504- 1515) as a consequence of the changes to the primary sequence of the PV capsid genes might contribute to the observed differences in efficiency of translation of the natural and modified PV capsid gene mRNAs in cultured cells. However, the enhancement of translation of the natural but not the modified mRNA that was observed after addition of tRNA in a mammalian in vitro translation system, which was not observed in a plant translation system, strengthens the argument that tRNA availability is rate limiting for translation of the natural gene in mammalian cells. A shortage of critical tRNAs could result in slowed elongation of the nascent peptide or premature termination of translation (Oba, et al., 1991, Biochimie 73:1109-1112). Slowed elongation appears to be the major consequence for the PV late gene.

Analysis of codon usage in the PV genome shows that PV late genes use many codons that mammalian cells rarely SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 use. For example, PV frequently uses UUA for leucine, CGU for arginine, ACA for threonine, and AUA for isoleucine, whereas these codons are significantly less often used in mammalian genes. In contrast, papillomavirus late genes can be expressed efficiently in yeast (Jansen, et al., 1995, Vaccine 13:1509-1514) (Sasagawa, et al., 1995, Virology 206:126-135) and the codon composition of yeast and papillomavirus genes are similar (Table An apparent exception is that PV L1 genes can be efficiently expressed in insect cells (Kirnbauer, et al., 1.992-, Proc. Natl. Acad. Sci.

USA 89:12180-12184) using recombinant baculovirus, or in various undifferentiated mammalian cells using recombinant vaccinia (Zhou, et al., 1991, Virology 185:251-257). As infection with vaccinia or baculovirus down regulates cellular protein synthesis, the efficient expression of the L1 capsid proteins under these circumstances may occur because less cellular mRNA is available in a virus infected cell to compete with the L1 mRNA for the rarer tRNAs.

Codon composition could be a more general determinant of gene expression within different stages of differentiation of the same tissue. Although the genetic code is essentially universal, different organisms show differences in codon composition of their genes, while the codon composition of genes tends to be relatively similar for all genes within each organism, and matched to the population of isotRNAs for that organism (Ikemura, 1981, J. Mol.

Biol. 146:1-21). However, populations of tRNAs in differentiating and neoplastic cells are different (Kanduc, 1997, Arch. Biochem. Biophys. 342:1-6; SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 Yang, and Comb, 1968, J. Mol. Biol. 31:138-142; Yang, and Novelli, 1968, Biochem. Biophys. Res. Commun. 31: 534-539) and the tRNA populations also vary in cells growing under different growth conditions (Doi, et al., 1968, J. Biol. Chem. 243:945-951). Accordingly, the inventors believe that codon composition and tRNA availability together provide a primitive mechanism for spatial and/or temporal regulation of gene expression. It is well recognized that the G+C content of many dsDNA viruses, a crude marker for viral gene codon composition, is markedly different from the G+C content of the DNA of the cells they infect (Strauss, et al., 1995, "Virus Evolution" in Virology (eds. Fields, B. et Lipipincott- Raven, Philadelphia, pp 153-171). Viruses may therefore have evolved to take advantage of codon composition to regulate their own program of gene expression, perhaps to avoid expression of lethal quantities of viral proteins in undifferentiated cells where the virus utilizes the cellular machinery to replicate its genome.

As the inventors' observations represent an apparently novel mechanism of regulation of gene translation within a single tissue, it is relevant to consider how this relates to previously proposed hypotheses for the restriction of expression of PV late genes to differentiated epithelium. A number of explanations have been proposed for the observation that PV late genes are only effectively expressed in differentiated epithelium. Reduced late gene transcription may reflect dependence of transcription from the late promoter on transcription factors SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 expressed only in differentiated epithelium, or may alternatively be due to suppression of late promoter transcription by viral (Stubenrauch, et al., 1996, J.

Virol. 70:119-126) or cellular gene products expressed in undifferentiated cells. The "late" promoters of HPV31b and of HPV5 (Haller, et al., 1995, Virology 214:245-255; Hummel, et al., 1992, J. Virol. 66:6070- 6080) are described as differentiation dependent, although the search for relevant transcription control factors in differentiated keratinocytes by conventional footprinting and DNA binding studies has to date been unrewarding. Our data show that capsid proteins are not translated from PV L1 and L2 mRNAs in cells transfected with CMV promoter-based expression vectors (Fig. suggesting that in addition to any transcriptional controls that may exist that there is a post-transcriptional block to capsid protein synthesis in undifferentiated cells. Sequences resembling 5' splice donor sites exist within L1 or L2 mRNA or within flanking untranslated message which are inhibitory to transcription of genes with which they are associated (Kennedy, et al., 1991, J. Virol.

65:2093-2097) (Furth, et al., 1994, Mol. Cell. Biol.

14:5278-5289). Other AU rich sequences in L1 or L2 mRNA promote mRNA degradation (Sokolowski, et al., 1997, Oncogene 15:2303-2319). These mechanisms inhibiting L1 and L2 expression in undifferentiated cells have yet to be shown to be inactive in differentiated epithelium, to explain the successful translation of late genes in this tissue.

Because inhibitory RNA sequences within the L1 coding sequence could have been rendered non- SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 functional by the systematic codon substitution employed in the experiments described herein and the untranslated inhibitory sequences were not included in the inventors' test system, the respective roles of inhibitory sequences and codon mismatch in suppression of PV late gene expression in cultured mammalian cells cannot be determined. However, regulatory sequences promoting RNA degradation or inhibiting translation are presumed to act through interaction with nuclear or cytoplasmic proteins (Sokolowski, et al., 1998, J.

Virol. 72:1504-1515), and inefficient translation of native sequence L1 mRNA was observed in a cell free translation system from anucleate cells, demonstrating that codon composition of the PV late genes must play some role in regulation of PV late gene translation.

Further evidence supporting the hypothesis that codon composition is an important determinant of PV capsid gene expression was gathered from an analysis of the 84 PV L1 sequences currently available in Genebank. The codon composition of the L1 genes, and particularly the frequency of usage of the rarer codons, was essentially the same across all the published sequences (data not shown) as would be predicted by the similar G+C content of the papillomavirus genomes. The PV L1 gene is relatively conserved at the amino acid level, showing 60 amino acid homology between PV genotypes, as might be expected by the constraints on capsid protein function. There are, however, no obvious constraining influences on the codon composition of the PV late genes beyond those of the inventors' hypothesis, as the late gene region does not code for other genes, SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 either in other reading frames or on the complementary DNA strand, and has no known cis acting regulatory functions. If codon composition of the capsid genes were not important for PV function, a considerable heterogeneity of codon usage might therefore be expected, given the evolutionary diversity of PVs (Chan, et al. 1995, J. Virol. 69:3074-3083).

Taken together, the data and evidence outlined herein makes a strong case that codon usage is a significant determinant of expression of PV late genes in undifferentiated and differentiated epithelial cells, and that this observation is generalizable. The relative role of message instability and codon mismatch in determining expression in differentiated tissues will require comparisons of transcriptional activity and translation of the L1 or L2 genes driven from strong constitutive promoters in differentiated and undifferentiated epithelium. Such work should now be feasible using either transgenic technology or keratinocyte raft cultures.

Although mechanisms of transcriptional regulation of PV L1 or L2 gene expression in the superficial layer of differentiated epithelium have been proposed (Zeltner et al., 1994, J. Virol.

68:3620; Brown, et al., 1995, Virology 214:259; Stoler et al., 1992, Hum. Pathol. 23:117; Hummel et al., 1995, J. Virol. 69:3381; Haller et al., 1995, Virology 214:245; Barksdale and Baker, 1993, J. Virol.

67:5605), measurable PV late gene mRNA is not always associated with production of late proteins (Zeltner et al., 1994, supra; Ozbun and Meyers, 1997,-J. Virol.

SUBSTITUTE SHEET (RULE 26) 67 71:5161), and the data presented here suggest that translation regulation may play a major part in controlling PV late gene expression. This observation has implications as herein described for the regulation of expression of genes related to the specialised functions of any differentiated tissue, and also for targeting of expression of therapeutic genes to such tissue while avoiding the potentially deleterious consequences of expression of the exogenous gene in a self renewing stem cell population.

The present invention has been described in terms of particular embodiments found or proposed by the present inventors to comprise preferred modes for the practice of the invention. Those of skill in the art will appreciate that, in light of the present disclosure, numerous modifications and changes may be made in the particular embodiments exemplified without departing from the scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

wn oQQ/fi.4Q PrrT/A I /nnIn TABLE LEGENDS TABLE 1 The codon usage data for human, cow yeast and wheat proteins are derived from published results(18). The BPV1 data are from the sequences in the Genbank database.

TABLE 2 Each iso-acceptor tRNA with anticodon shown as superscript are shown on top row. The indicates the abundance of tRNA wherein each indicates about 10 fold increase.

SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 TABLE -1 Frequency (per one organia.sms.

thousand) of codon usage for individual Amino acids

ARG

LEU

SER

THR

Codons Human

CGA

CGC

CGG

CGU

AGA

AGG

CUA

CUC

CUG

CUU

UUA

UUG

TJCA

Ucc

UCG

UCU

AGC

AGU

ACA

ACC

ACG

ACU

5.4 11.3 10.4 4.7 9.9 11.1 6.2 19.9 42.5 10.7 5.3 11.0 9.3 17 .7 4.2 13.2 18 .7 9.4 14 .4 23.0 6.7 12 .7 Cow 5.5 12.2 11.2 3.7 9.9 11.4 4.9 21.2 46.6 10.6 4.0 9.6 7.6 17.6 4.5 11.2 18.7 8.6 11.4 21.1 7.8 9.6 Yeast Wheat BPVL1/ L2 2.3 2.0 1.1 7.5 24. 0 7.5 11.8 4.1 8.3 9.6 24. 5 32 .1 15. 6 14.4 6.5 24 .6 7.1 11. 7 15. 6 13. 9 6.7 22 .0 2.3 7.5 4.6 1.1 4.1 7.1 12 .1 18.6 15 .5 6.5 1.8 15 .3 14 .6 10.1 9.6 14 .8 12 .8 12 .9 4.6 15 .9 4.5 11. 8 7.2 4.1 5.1 10.4 14.4 9.3 18.6 6.2 15.5 20.7 14 15.5 16.6 11.4 6.2 15 12 .4 21.7 37.3 19 .7 4.1 28.0 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 Amino Codons Human Cow Yeast Wheat acids

PRO

ALA

GLY

VAL

LYS

ASN

GLN

HIS

GILU

AS P

CCA

CCC

CCG

CCU

GCA

GCC

GCG

GCU

GGA

GGC

GGG

GGU

GUA

GUC

GUG

GUU

AAA

AAG

AAC

AAU

CAA

CAG

CAC

CAU

GAA

GAG

GAC

GAU

14 .6 20.0 6.5 15.5 14. 0 29.1 7.2 19. 6 17.1 25.4 17.3 11.2 5.9 16.3 30.9 10.4 22.2 34 .9 22 .6 16.6 11.1 33.6 14 .2 9.3 26.8 41.4 29.0 21.7 12 .0 19.2 7.9 14 .6 13 .1 35.8 9.3 19.1 16.2 28.1 19.2 11.8 5.1 18.4 32.9 9.9 21.6 37.1 22.4 12 .5 9.7 34.4 14 .0 7.5 24 .4 45.4 31.5 19 .2 21.4 5.9 4.1 12 .8 15.3 15.5 5.1 28.3 8.9 8.9 5.1 34 .9 10.0 14 .9 9.5 26.6 37.7 35.2 25. 8 31.4 29.8 10.4 8.2 12 .3 48. 9 16. 9 22. 3 37. 0 71.2 11. 1 19.4 10 .3 11.2 19.5 13 .8 9.6 25.9 28.0 28.5 9.6 4.4 14 .8 12 .9 11 .6 4.5 17.4 14.2 6.7 171 .8 79.4 8.2 7.1 7.8 19.7 13 .0 4.0 BPVL1 L2 22 .8 15 0.0 33.1 33.1 17.6 4.1 13.5 22.8 12 .4 22.8 18.6 15.5 6.2 23.8 16 .6 37.2 13 10.3 24.8 22 .8 17.6 6.2 13 .4 36.2 21.7 18.6 33.1 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 Amino Codons acids Human Cow- Yeast Wheat

TYR

CYS

PHE

ILE

UAC

UAU

UGC

TJGU

TUtc UUtJ

AUA

AUC

ATUE

18. 8 12 .5 14 .5 9.9 22 .6 15 .8 5.8 24 .3 14 .9 20.3 10.5 13 .9 9.4 25.5 17 .0 5.2 25.8 13 .1 16.5 16.5 3.7 7.6 20.0 23.2 12 .8 18.4 31.1.

24.5 12 .5 14.8 4.9 14 .1 15 .0 5.4 19.7 10.7 BPVL1 L2 17 .6 18 .6 5.2 5.2 7.2 23.8 22.7 8.2 20.7 SUBSTITUTE SHEET (RULE 26) TABLE 2 tRNA population changes as KC starts to differentiate.

tRNA ArgcGA Ala Gc Hi sC LeUC LeuCT LysAAG LysA-" Met IiProcc Supra Basal tRNA Val1GTA Va 1GTI His'cC AsnAAc ThrACl Met"-o GlYGGA Supra Basal EDITORIAL NOTE NO. 81999/98 This specification contains a sequence listing following the description and is numbered as follows: Sequence listing pages 1 19 Claim pages 73 89 WO 99/02694 PCT/AU98/00530 SEQUENCE LISTING <110> The University of Queensland <120> NUCLEIC ACID SEQUENCE AND METHOD FOR SELECTIVELY EXPRESSING A PROTEIN IN A TARGET CELL OR TISSUE <130> Selective Expression <140> PCT/AU98/00530 <141> 1998-07-09 <150> PO7765 <151> 1997-07-09 <150> PO9467 <151> 1997-09-11 <160> 34 <170> PatentIn Ver. <210> 1 <211> 1488 <212> DNA <213> Bovine papillomavirus type 1 <220> <221> CDS <222> (1)..(1488) <220> <223> L1 open reading frame (wild-type) <400> 1 atg gcg ttg tgg caa caa ggc cag aag Met Ala Leu Trp Gin Gin Gly Gin Lys 1 5 gta agc aag gtg ctt tgc agt gaa acc Val Ser Lys Val Leu Cys Ser Glu Thr 25 ctg Leu 10 tat ctc cct cca Tyr Leu Pro Pro acc cct Thr Pro tat gtg caa aga Tyr Val Gin Arg aaa age att Lys Ser lie cat cca tat His Pro Tyr ttt tat cat Phe Tyr His gca gaa acg gag Ala Glu Thr Glu cgc Arg ctg cta act ata Leu Leu Thr Ile 144 tac cca Tyr Pro gtg tct ate ggg Val Ser Ile Gly gcc Ala 55 aaa act gtt cct Lys Thr Val Pro aag Lys gtc tct gca aat Val Ser Ala Asn cag Gin tat agg gta ttt Tyr Arg Val Phe aaa Lys 70 ata caa cta cct Ile Gin Leu Pro ccc aat caa ttt Pro Asn Gin Phe gca Ala SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 cta Leu cca Pro gt a Val aat Asn cta Leu 145 gaa Glu gaa Giu gat Asp att Ile tgc Cys 225 agc Ser tgg Trp, tta Leu cct Pro gtc Val act Thr aga Arg 130 gat Asp ggg Gly aat Asn ggg Gly aat Asn 210 ttg Leu atg Met ac c Thr aag Lys gac Asp ata Ile ggg Gly 115 aaa Lys gct Ala gaa Giu Gly gat Asp 195 gca Al a tac Tyr ttc Phe aga Arg aat Asn 275 agg Arg ggt Gly 100 cac His gt c Val aag Lys tat Tyr gcc Ala 180 atg Met agt Ser cca Pro ttt Phe ggg Gly 260 aat Asn act Thr gtg Val ccc Pro acc Thr caa Gin tgg Trp 165 tgc Cys atg Met aaa Lys gac Asp ttt Phe 245 ggc Gly aaa Lys gt t Val cag Gin act Thr acc Thr caa Gin 150 aca Thr cct Pro gaa Giu tca Ser tac Tyr 230 gca Ala tcg Ser ggg Gly cac His gtg Val ttt Phe c aa Gin 135 cag Gin aca Thr cct Pro att Ile gat Asp 215 ctc Leu agg Arg gag Glu gat Asp aac Asn tcc Ser aat Asn 120 aca Thr att Ile gcc Ala ctt Leu ggg Giy 200 cta Leu aaa Lys aaa Lys aaa Lys gcc Ala 280 cca Pro aga Arg 105 gct Ala aca Thr ctg Leu cgt Arg gaa Giu 185 ttt Phe cct Pro atg Met gaa Glu gaa Giu 265 acc Thr agt Ser 90 ggg Gly ttg Leu gat Asp ttg Leu cca Pro 170 tta Leu ggt Giy ctt Leu gct Al a cag Gin 250 gcc Ala ctt Leu aa a Lys cag Gin ctt.

Leu gac Asp cta Leu 155 tgt Cys aaa Lys gca Al a gac Asp gag Glu 235 gtg Val cct Pro aaa Lys cct ctt gga Pro Leu Gly 110 gat Asp agg Arg 140 ggc Gly gtt Val1 aac Asn gcc Ala att Ile 220 gac Asp tat Tyr acc Thr ata Ile gat Asp 300 gag cgg ctg gtg tgg Giu Arg Leu Val Trp gca Ala 125 aaa Lys tgt Cys act Thr aag Lys aac Asn 205 c aa Gin gct Ala gtt Val aca Thr Ccc Pro 285 gaa Glu caa Gin acc Thr gat Asp c ac His 190 tt c Phe aat Asn gct Ala aga Arg gat Asp 270 agt Ser ggt Gly aat Asn aca Thr cct Pro cgt Arg 175 ata Ile aaa Lys gag Giu ggt Gly c ac His 255 ttt Phe gtg Val act Thr gtg Vai Gly gct Al a 160 cta Leu gaa Glu gaa Giu atc Ile aat As n 240 atc Ile tat Tyr cat His ttt ggt agt ccc agt ggc tca cta gtc tca act Phe Gly Ser Pro Ser Gly Ser Leu Val Ser Thr aat caa att ttt Asn Gin Ile Phe 290 295 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 cgg ccc tac tgg Arg Pro Tyr Trp cta Leu 310 ttc cgt gcc cag Phe Arg Ala Gin ggc Gly 315 atg aac aat gga Met Asn Asn Gly att Ile 320 960 1008 gca tgg aat aat Ala Trp Asn Asn tta Leu 325 ttg ttt tta aca Leu Phe Leu Thr gtg Val 330 ggg gac aat aca Gly Asp Asn Thr cgt ggt Arg Giy 335 act aat ctt Thr Asn Leu tat gat 'agc Tyr Asp Ser 355 acc Thr 340 ata agt gta gcc Ile Ser Val Ala gat gga acc cca Asp Gly Thr Pro cta aca gag Leu Thr Giu 350 gaa gaa tat Giu Giu Tyr 1056 1104 tca aaa ttc aat Ser Lys Phe Asn gta Val 360 tac cat aga cat Tyr His Arg His aag cta Lys Leu 370 gcc ttt ata tta Ala Phe Ile Leu cta tgc tct gtg Leu Cys Ser Val gaa Giu 380 atc aca gct caa Ile Thr Ala Gin gtg tca cat ctg Vai Ser His Leu caa Gin 390 gga ctt atg ccc Gly Leu Met Pro tct Ser 395 gtg ctt gaa aat Vai Leu Giu Asn tgg Trp 400 1152 1200 1248 1296 gaa ata ggt gtg Giu Ile Gly Val cag Gin 405 cct cct acc tca Pro Pro Thr Ser ata tta gag gac Ile Leu Giu Asp acc tat Thr Tyr 415 cgc tat ata Arg Tyr Ile gca aaa gaa Ala Lys Giu 435 tct cct gca act Ser Pro Ala Thr aaa Lys 425 tgt gca agc aat gta att cct Cys Ala Ser Asn Val Ile Pro 430 gac cct tat gca Asp Pro Tyr Ala ggg Gly 440 ttt aag ttt tgg Phe Lys Phe Trp aac Asn 445 ata gat ctt Ile Asp Leu 1344 aaa gaa Lys Giu 450 aag ctt tct ttg Lys Leu Ser Leu gac Asp 455 tta gat caa ttt Leu Asp Gin Phe ccc Pro 460 ttg gga aga aga Leu Gly Arg Arg tta gca cag caa Leu Ala Gin Gin ggg Gly 470 gca gga tgt tca Ala Giy Cys Ser act Thr 475 gtg aga aaa cga Val Arg Lys Arg aga Arg 480 1392 1440 1488 att agc caa aaa Ile Ser Gin Lys act Thr 485 tcC agt aag cct Ser Ser Lys Pro aaa aaa aaa aaa Lys Lys Lys Lys aaa taa Lys 495 <210> 2 <211> 495 <212> PRT <213> Bovine papiliomavirus type 1 <400> 2 Met Ala Leu Trp Gin Gin Gly Gin Lys Leu 1 5 10 Tyr Leu Pro Pro Thr Pro SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 Val1 Phe Tyr Gin Leu Pro Val Asn Leu 145 Glu Giu Asp Ile Cys 225 Ser Trp Leu Phe Asn 305 Ser Tyr Pro Tyr Pro Vai Thr Arg 130 Asp Gly Asn Gly Asn 210 Leu Met Thr Lys Gly 290 Arg Lys His Val Arg Asp Ile Giy 115 Lys Al a Glu Gly Asp 195 Ala Tyr Phe Arg As n 275 Ser Pro Vai Al a Ser Vai Arg Gly 100 His Val1 Lys Tyr Ala 180 Met Ser Pro Phe Gly 260 Asn Pro Tyr Leu Cys Ser Giu Giu Ile Phe Thr Val Pro Thr Gin Trp 165 Cys Met Lys Asp Phe 245 Gly Lys Ser Trp Thr Giy Lys 70 Val Gin Thr Thr Gin 150 Thr Pro Giu Ser Tyr 230 Ala Ser Gly Gly Leu 310 Giu Al a Ile His Val Phe Gin 135 Gin Thr Pro Ile Asp 215 Leu Arg Glu Asp Ser 295 Phe Arg Lys Gin Asn Ser As n 120 Thr Ile Ala Leu Gly 200 Leu Lys Lys Lys Ala 280 Leu Arg Thr Tyr Val 25 Leu Leu Thr Thr Val Pro Leu Pro Asp 75 Pro Ser Lys 90 Arg Gly Gin 105 Ala Leu Leu Thr Asp Asp Leu Leu Leu 155 Arg Pro Cys 170 Giu Leu Lys 185 Phe Gly Ala Pro Leu Asp Met Ala Giu 235 Giu Gin Val 250 Giu Ala Pro 265 Thr Leu Lys Val Ser Thr Ala Gin Gly 315 Gin Ile Lys Pro Giu Pro Asp Arg 140 Gly Val Asn Ala Ile 220 Asp Tyr Thr Ile Asp 300 Met Lys His Ser Gin Leu Gly 110 Glu Gin Thr Asp His 190 Phe Asn Ala Arg Asp 270 Ser Gin Asn Ser Pro Al a Phe Val Gly Asn Thr Pro Arg 175 Ile Lys Glu Gly His 255 Phe Val1 Ile Gly Ile Tyr Asn Al a Trp Thr Val Gly Ala 160 Leu Giu Giu Ile Asn 240 Ile Tyr His Phe Ile 320 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530

V

Ala Trp Asn Asn Leu Leu Phe Leu Thr Val Gly Asp Asn Thr Arg Gly 325 330 335 Thr Asn Leu Thr Ile Ser Val Ala Ser Asp Gly Thr Pro Leu Thr Glu 340 345 350 Tyr Asp Ser Ser Lys Phe Asn Val Tyr His Arg His Met Glu Glu Tyr 355 360 365 Lys Leu Ala Phe Ile Leu Glu Leu Cys Ser Val Glu lie Thr Ala Gin 370 375 380 Thr Val Ser His Leu Gin Gly Leu Met Pro Ser Val Leu Glu Asn Trp 385 390 395 400 Glu Ile Gly Val Gin Pro Pro Thr Ser Ser lie Leu Glu Asp Thr Tyr 405 410 415 Arg Tyr Ile Glu Ser Pro Ala Thr Lys Cys Ala Ser Asn Val Ile Pro 420 425 430 Ala Lys Glu Asp Pro Tyr Ala Gly Phe Lys Phe Trp Asn Ile Asp Leu 435 440 445 Lys Glu Lys Leu Ser Leu Asp Leu Asp Gin Phe Pro Leu Gly Arg Arg 450 455 460 Phe Leu Ala Gin Gin Gly Ala Gly Cys Ser Thr Val Arg Lys Arg Arg 465 470 475 480 lie Ser Gin Lys Thr Ser Ser Lys Pro Ala Lys Lys Lys Lys Lys 485 490 495 <210> 3 <211> 1488 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1)..(1488) <220> <223> Description of Artificial Sequence: Bovine papillomavirus type 1 L1 open reading frame (humanized) <220> <223> Wild-type codons replaced with synonymous codons used at relatively high frequency by human genes <400> 3 atg gcc ctg tgg cag cag ggc cag aag ctg tac ctg ccc cct acc ccc 48 Met Ala Leu Trp Gin Gin Gly Gin Lys Leu Tyr Leu Pro Pro Thr Pro 1 5 10 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 gtg agc aag gtg ctt tgc agt gaa acc tat gtg caa aga aaa agc att 96 Val Ser Lys Val Leu Cys Ser Giu Thr Tyr Val Gin Arg Lys Ser Ile 25 ttt tat cat gca gaa acg gag cgc ctg ctg acc atc gga cac ccc tat 144 Phe Tyr His Ala Glu Thr Giu Arg Leu Leu Thr Ile Gly His Pro Tyr 40 tac ccc gtg tcc atc ggg gcc aag act gtg cct aag gtg tcc gcc aat 192 Tyr Pro Val Ser Ile Gly Ala Lys Thr Val Pro Lys Val Ser Ala Asn 55 cag tat agg gtg ttc aaa atc caa Ctg cct gat ccc aat caa ttt gca 240 Gin Tyr Arg Val Phe Lys Ile Gin Leu Pro Asp Pro Asn Gin Phe Ala 70 75 ctg cct gac agg acc gtg cac aac ccc agc aaa gag cgg ctg gtg tgg 288 Leu Pro Asp Arg Thr Val His Asn Pro Ser Lys Giu Arg Leu Val Trp 90 cca gtg atc ggc gtg cag gtg tcc aga ggc cag cct ctg ggc ggc acc 336 Pro Val Ile Gly Val Gin Vai Ser Arg Gly Gin Pro Leu Gly Gly Thr 100 105 110 gtg act ggg cac ccc act ttt aat gct ttg ctt gat gca gaa aat gtg 384 Val Thr G-iy His Pro Thr Phe Asn Ala Leu Leu Asp Ala Glu Asn Val 115 120 125 aat aga aaa gtc acc acc cag acc acc gac gac agg aaa cag aca ggc 432 Asn Arg Lys Val Thr Thr Gin Thr Thr Asp Asp Arg Lys Gin Thr Gly 130 135 140 ctg gat gcc aag cag cag cag atc ctg ctg ctg ggc tgt acc cct gct 480 Leu Asp Ala Lys Gin Gin Gin Ile Leu Leu Leu Gly Cys Thr Pro Ala 145 150 155 160 gaa ggg gaa tat tgg aca aca gcc cgt cca tgt gtg acc gac cgt cta 528 Glu Gly Giu Tyr Trp Thr Thr Ala Arg Pro Cys Vai Thr Asp Arg Leu 165 170 175 gaa aac ggc gcc tgc cct cct ctg gag ctg aaa aac aag cac atc gaa 576 Glu Asn Gly Ala Cys Pro Pro Leu Giu Leu Lys Asn Lys His Ile Glu 180 185 190 gat ggg gat atg atg gaa att ggg ttt ggt gca gcc aac ttc aaa gaa 624 Asp Gly Asp Met Met Giu Ile Gly Phe Gly Ala Ala Asn Phe Lys Glu 195 200 205 att aat gca agt aaa tca gat cta cct ctg gac atc caa aat gag atc 672 Ile Asn Aia Ser Lys Ser Asp Leu Pro Leu Asp Ile Gin Asn Giu Ile 210 215 220 tgc ctg tac ccc gac tac ctg aaa atg gct gag gac gcc gcc ggc aac 720 Cys Leu Tyr Pro Asp Tyr Leu Lys Met Ala Giu Asp Ala Ala Gly Asn 225 230 235 240 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 agc atg ttc ttc Ser Met Phe Phe ttc Phe 245 gcc agg aag gag Ala Arg Lys Glu c ag Gin 250 gtg tac gtg aga.

Val Tyr Val Arg cac atc His Ile 255 tgg acc aga Trp Thr Arg ttg aag aac Leu Lys Asn 275 ggc Gly 260 ggc tcc gag aaa Giy Ser Glu Lys gaa Giu 265 gcc cct acc aca Ala Pro Thr Thr gat ttt tat Asp Phe Tyr 270 agc gtg cac Ser Vai His aac aag ggc gac Asn Lys Giy Asp gcc Al a 280 acc ctg aag atc Thr Leu Lys Ile ttc ggc Phe Gly 290 agc ccc agc ggc Ser Pro Ser Giy cta gtg tcc acc Leu Val Ser Thr gac Asp 300 aac cag atc ttc Asn Gin Ile Phe aac Asn 305 cgg ccc tac tgg Arg Pro Tyr Trp ctg Leu 310 ttc cgc gcc cag Phe Arg Ala Gin ggc Giy 315 atg aac aat gga Met Asn Asn Gly at t Ile 320 912 960 1008 gcc tgg aac aac Aia Trp Asn Asn ctg Leu 325 ctg ttc ctg acc Leu Phe Leu Thr gtg Val 330 ggc gac aac aca.

Giy Asp Asn Thr cgt ggc Arg Gly 335 acc aac ctg Thr Asn Leu tat gat agc Tyr Asp Ser 355 acc Thr 340 atc agc gtg 9CC Ile Ser Val Ala tcc Ser 345 gat gga. acc cca Asp Gly Thr Pro ctg acc gag Leu Thr Giu 350 gag gag tat Giu Giu Tyr 1056 1104 tcg aaa ttc aac Ser Lys Phe Asn gtg Val1 360 tac cac aga. cac Tyr His Arg His aag cta Lys Leu 370 gcc ttc atc ctg Ala Phe Ile Leu ctg tgc tcc gtg Leu Cys Ser Vai gag Giu 380 atc acc gcc cag Ile Thr Ala Gin acc Thr 385 gtg tcc cat ctg Val Ser His Leu caa Gin 390 gga ctg atg CCC Gly Leu Met Pro tcc Ser 395 gtg ctg gag aat Val Leu Giu Asn tgg Trp 400 1152 1200 1248 gag atc ggc gtg Giu Ile Giy Vai cag Gin 405 ccc ccc acc tca Pro Pro Thr Ser t cg Ser 410 atc: ttg gag gac Ile Leu Giu Asp acc tac Thr Tyr 415 cgc tac atc Arg Tyr Ile gca aaa gaa Ala Lys Giu 435 gag Giu 420 tcc ccc gcc acc Ser Pro Ala Thr tgt gcc agc aac Cys Ala Ser Asn gtg att cct Vai Ile Pro 430 atc gac ctg Ile Asp Leu gac cct tat gca Asp Pro Tyr Aia ggg Gly 440 ttt aag ttc tgg Phe Lys Phe Trp 1296 1344 1392 aag gag Lys Giu 450 aag ctg tct ctg gac ctg gat cag ttc ccc ttg ggc aga. aga.

Lys Leu Ser Leu Asp Leu Asp Gin Phe Pro Leu Gly Arg Arg 455 460 SUBSTITUTE SHEET (RULE 26) Wn OO/A'VKOA PCT/.&IIQR/00530 ~A1C OOfl~~OA CT/ITQ /flf~flVT ~TS.UZviii ttt ctg gcc cag cag ggg gcc ggc tgt tcc acc gtg aga aaa cgc agg 1440 Phe Leu Ala Gin Gin Gly Ala Gly Cys Ser Thr Val Arg Lys Arg Arg 465 470 475 480 atc agc cag aag acc tcc agc aag ccc gcc aag aag aag aaa aag taa 1488 Ile Ser Gin Lys Thr Ser Ser Lys Pro Ala Lys Lys Lys Lys Lys 485 490 495 <210> 4 <211> 495 <212> PRT <213> Artificial Sequence <400> 4 Met Aia Leu Trp Gin Gin Gly Gin Lys Leu Tyr Leu Pro Pro Thr Pro 1 5 10 Val Ser Lys Val Leu Cys Ser Glu Thr Tyr Val Gin Arg Lys Ser Ile 25 Phe Tyr His Ala Giu Thr Glu Arg Leu Leu Thr Ile Gly His Pro Tyr 40 Tyr Pro Val Ser Ile Gly Ala Lys Thr Val Pro Lys Val Ser Ala Asn 55 Gin Tyr Arg Val Phe Lys Ile Gin Leu Pro Asp Pro Asn Gin Phe Ala 70 75 Leu Pro Asp Arg Thr Vai His Asn Pro Ser Lys Glu Arg Leu Val Trp 90 Pro Val Ile Gly Val Gin Val Ser Arg Gly Gin Pro Leu Gly Gly Thr 100 105 110 Val Thr Giy His Pro Thr Phe Asn Ala Leu Leu Asp Ala Glu Asn Val 115 120 125 Asn Arg Lys Val Thr Thr Gin Thr Thr Asp Asp Arg Lys Gin Thr Gly 130 135 140 Leu Asp Ala Lys Gin Gin Gin Ile Leu Leu Leu Gly Cys Thr Pro Ala 145 150 155 160 Glu Gly Giu Tyr Trp, Thr Thr Ala Arg Pro Cys Val Thr Asp Arg Leu 165 170 175 Giu Asn Gly Ala Cys Pro Pro Leu Glu Leu Lys Asn Lys His Ile Glu 180 185 190 Asp Gly Asp Met Met Glu Ile Gly Phe Gly Ala Ala Asn Phe Lys Glu 195 200 205 Ile Asn Ala Ser Lys Ser Asp Leu Pro Leu Asp Ile Gin Asn Giu Ile 210 215 220 SUBSTITUTE SHEET (RULE 26) WO 00/'9OA VPC T/AU98/00530 ix Cys Leu Tyr Pro Asp Tyr Leu Lys Met Ala Glu Asp Ala Ala Gly Asn 225 230 235 240 Ser Met Phe Phe Phe Ala Arg Lys Glu Gin Val Tyr Val Arg His Ile 245 250 255 Trp Thr Arg Gly Gly Ser Glu Lys Glu Ala Pro Thr Thr Asp Phe Tyr 260 265 270 Leu Lys Asn Asn Lys Gly Asp Ala Thr Leu Lys Ile Pro Ser Val His 275 280 285 Phe Gly Ser Pro Ser Gly Ser Leu Val Ser Thr Asp Asn Gin Ile Phe 290 295 300 Asn Arg Pro Tyr Trp Leu Phe Arg Ala Gin Gly Met Asn Asn Gly Ile 305 310 315 320 Ala Trp Asn Asn Leu Leu Phe Leu Thr Val Gly Asp Asn Thr Arg Gly 325 330 335 Thr Asn Leu Thr Ile Ser Val Ala Ser Asp Gly Thr Pro Leu Thr Glu 340 345 350 Tyr Asp Ser Ser Lys Phe Asn Val Tyr His Arg His Met Glu Glu Tyr 355 360 365 Lys Leu Ala Phe Ile Leu Glu Leu Cys Ser Val Glu Ile Thr Ala Gin 370 375 380 Thr Val Ser His Leu Gin Gly Leu Met Pro Ser Val Leu Glu Asn Trp 385 390 395 400 Glu Ile Gly Val Gin Pro Pro Thr Ser Ser Ile Leu Glu Asp Thr Tyr 405 410 415 Arg Tyr Ile Glu Ser Pro Ala Thr Lys Cys Ala Ser Asn Val Ile Pro 420 425 430 Ala Lys Glu Asp Pro Tyr Ala Gly Phe Lys Phe Trp Asn Ile Asp Leu 435 440 445 Lys Glu Lys Leu Ser Leu Asp Leu Asp Gin Phe Pro Leu Gly Arg Arg 450 455 460 Phe Leu Ala Gin Gin Gly Ala Gly Cys Ser Thr Val Arg Lys Arg Arg 465 470 475 480 Ile Ser Gin Lys Thr Ser Ser Lys Pro Ala Lys Lys Lys Lys Lys 485 490 495 <210> <211> 1410 <212> DNA <213> Bovine papillomavirus type 1 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 <220> <221> CDS <222> (1410) <220> <223> L2 open reading frame (wild-type) <400> atg Met 1 agg Arg gta Val gca Ala gtg Val.

tcc Ser acc Thr ctt Leu gcc Ala gat Asp 145 ctg Leu ccc Pro agt gca Ser Ala acc tgc Thr Cys gaa gga Giu Gly atc tac Ile Tyr gcc gca Ala Ala aca tca Thr Ser cga Arg aag Lys gat Asp tta Leu ggt Gly tcg Ser agt Ser 100 ttg Leu ata Ile tcc Ser cct Pro cgt Arg aaa Lys 5 caa Gln act Thr gga Gly gga Gly ctt Leu ata Ile cgt Arg gtc Val ata Ile gag Glu 165 cca Pro aga Arg gcg Al a ata Ile ggg Gly tca Ser 70 gca Al a ggt Gly cca Pro act Thr ggt Gly 150 ggt Gly act Thr gta aaa Val Lys ggc aca Gly Thr gca gat Ala Asp 40 cta gga Leu Gly 55 cca agg Pro Arg tca ata Ser Ile gcg ggc Ala Gly ggg gtg Gly Val 120 cct gat Pro Asp 135 aca. gac Thr Asp ccc gag Pro Glu tgg caa Trp Gln cgt Arg tgt Cys 25 aaa Lys ata Ile tac Tyr gga Gly att Ile 105 tat Tyr gct Ala tcg Ser gac Asp gta Val 185 gcc Ala cca Pro att Ile gga Gly aca Thr tcc Ser cct Pro gag Giu gtt Val tcc Ser ata Ile 170 agc Ser agt gcc Ser Ala cca gat Pro Asp ttg aaa Leu Lys aca tgg Thr Trp cca ctc Pro Leu aga gct Arg Ala tta gac Leu Asp gac act Asp Thr cct gca Pro Ala 140 acg gag Thr Glu 155 gcg gtt Ala Val aat gct tat Tyr gtg Val ttt Phe tct Ser cga Arg gt a Val acc Thr gtg Val 125 gat Asp acc Thr ctt Leu gtt gac Asp ata Ile ggg Gly act Thr aca Thr aca Thr ctt Leu 110 cta Leu t ca Ser ctc Leu gag Giu cat 48 96 144 192 24.0 288 336 384 432 480 528 576 cgc Arg ggg Giy cct Pro 130 gcc Ala cta Leu ctg Leu ccc Pro gcc Ala 115 gca Ala ctg Leu gag Giu gac Asp Asn Ala Val His Gin Ser SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 tct gca tac Ser Ala Tyr 195 cac gcc cci cig His Ala Pro Leu cag Gin 200 cig caa tcg icc Leu Gin Ser Ser gca gaa aca Ala Giu Thr 624 tct ggt Ser Gly 210 tta gaa aat aii Leu Giu Asn Ile tt Phe 215 gta gga ggc tcg Val Gly Gly Ser ggt Gly 220 ita ggg gat aca Leu Giy Asp Thr gga Gly 225 gga gaa aac at Gly Giu Asn Ile ctg aca tac tic Leu Thr Tyr Phe ggg Giy 235 icc cca cga aca Ser Pro Arg Thr agc Ser 240 672 720 768 acg ccc cgc agi Thr Pro Arg Ser ati Ile 245 gcc ici aaa tca Ala Ser Lys Ser cgi Arg 250 ggc aii ita aac Gly Ile Leu Asn igg tic Trp Phe 255 agt aaa cgg Ser Lys Arg ica icc caa Ser Ser Gin 275 tac Tyr 260 tac aca cag gig Tyr Thr Gin Val acg gaa gai cci Thr Giu Asp Pro gaa gig iii Giu Vai Phe 270 cca gci gig Pro Ala Val 816 864 aca iii gca aac Thr Phe Ala Asn cca Pro 280 cig iai gaa gca Leu Tyr Giu Aia cii aag Leu Lys 290 gga cci agi gga Giy Pro Ser Gly cgi Arg 295 gii gga cic agi Val Gly Leu Ser cag Gin 300 gii tat aaa cci Val Tyr Lys Pro gat Asp 305 aca cii aca aca Thr Leu Thr Thr agc ggg aca gag Ser Giy Thr Giu gig Vai 315 gga cca cag cia Giy Pro Gin Leu 912 960 1008 gic agg tac ica Val Arg Tyr Ser tig Leu 325 agi act ata cat Ser Thr Ile His gaa Giu 330 gat gia gaa gca Asp Val Giu Ala aic ccc Ile Pro 335 tac aca gt Tyr Thr Val gaa gag caa Giu Glu Gin 355 gat Asp 340 gaa aai aca cag Giu Asn Thr Gin cii gca tic gia Leu Ala Phe Val ccc tig cai Pro Leu His 350 iii agi gag Phe Ser Giu 1056 1104 gca ggi iii gag Ala Gly Phe Giu gag Giu 360 ata gaa ita gat Ile Glu Leu Asp aca cat Thr His 370 aga cig cia cci Arg Leu Leu Pro aac acc ici ict Asn Thr Ser Ser aca Thr 380 cci git ggi agi Pro Val Giy Ser gia cga aga agc Vai Arg Arg Ser cic Leu 390 ait cca act cga Ile Pro Thr Arg iii agi gca aca Phe Ser Ala Thr cgg Arg 400 1152 1200 1248 cci aca ggt gt Pro Thr Gly Val gia Vai 405 acc tat ggc ica Thr Tyr Gly Ser cci Pro 410 gac act tac ict Asp Thr Tyr Ser gci agc Ala Ser 415 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 cca gtt act gac cct gat tct acc tc: Pro Val Thr Asp Pro Asp Ser Thr Se 420 42 act act act aca cca atc att ata at Thr Thr Thr Thr Pro Ile Ile Ile Ii 435 440 tac agc agt aac tac acc ttg cat cc Tyr Ser Ser Asn Tyr Thr Leu His Pr 450 455 aaa cgg aaa cat gcc taa Lys Arg Lys His Ala 465 470 <210> 6 <211> 469 <212> PRT <213> Bovine papillomavirus type 1 xii t cct agt r Pro Ser 5 t gat ggg e Asp Gly c tcc ttg o Ser Leu cta gtt atc gat gac Leu Val Ile Asp Asp 430 cac aca gtt gat ttg His Thr Val Asp Leu 445 ttg agg aaa cga aaa Leu Arg Lys Arg Lys 460 1296 1344 1392 1410 <400> 6 Met Ser Ala Arg Lys Arg Val 1 Arg Val Ala Val Ser Thr Leu Al a Asp 145 Leu Thr Glu Ile Ala Thr Arg Gly Pro 130 Ala Leu Cys Gly Tyr Al a Ser Pro Ala 115 Ala Leu Glu Lys Asp Leu Gly Ser Ser 100 Leu Ile Ser Pro 5 Gin Thr Gly Gly Leu Ile Arg Val Ile Glu 165 Ala Ile Gly Ser 70 Al a Gly Pro Thr Gly 150 Gly Gly Aia Leu Pro Ser Ala Gly Pro 135 Thr Pro Lys Thr Asp 40 Gly Arg Ile Gly Val 120 Asp Asp Glu Arg Cys 25 Lys Ile Tyr Gly Ile 105 Tyr Al a Ser Asp Ala 10 Pro Ile Gly Thr Ser 90 Pro Glu Val Ser Ile 170 Ser Pro Leu Thr Pro 75 Arg Leu Asp Pro Thr 155 Ala Tyr Val1 Phe Ser Arg Val Thr Val1 125 Asp Thr Leu Asp Ile Gly Thr Thr Thr Leu 110 Leu Ser Leu Glu Leu Arg Gly Gly Al a Ala Glu Pro Gly Ile Leu 175 Tyr Lys Leu Arg Gly Gly Thr Glu Leu Thr 160 Gin SUBSTITUTE SHEET (RULE 26) Wn 001fl')AGA Df, TIAU98/00530 U~d~OO~f)~OADC TAU980053 Pro Leu Asp Arg Pro Thr Trp Gin Val Ser Asn Ala Val His Gin Ser 180 185 190 Ser Ala Tyr His Ala Pro Leu Gin Leu Gin Ser Ser Ile Ala Glu Thr 195 200 205 Ser Gly Leu Giu Asn Ile Phe. Val Gly Giy Ser Gly Leu Gly Asp Thr 210 215 220.

Gly Giy Giu Asn Ile Glu Leu Thr Tyr Phe Gly Ser Pro Arg Thr Ser 225 230 235 240 Thr Pro Arg Ser Ile Ala Ser Lys Ser Arg Gly Ile Leu Asn Trp Phe 245 250 255 Ser Lys Arg Tyr Tyr Thr Gin Val Pro Thr Giu Asp Pro Giu Val Phe 260 265 270 Ser Ser Gin Thr Phe Ala Asn Pro Leu Tyr Giu Ala Glu Pro Ala Vai 275 280 285 Leu Lys Gly Pro Ser Giy Arg Val Gly Leu Ser Gin Vai Tyr Lys Pro 290 295 300 Asp Thr Leu Thr Thr Arg Ser Giy Thr Giu Vai Gly Pro Gin Leu His 305 310 315 320 Vai Arg Tyr Ser Leu Ser Thr Ile His Giu Asp Val Giu Ala Ile Pro 325 330 335 Tyr Thr Val Asp Glu Asn Thr Gin Giy Leu Ala Phe Val Pro Leu His 340 345 350 Giu Giu Gin Ala Giy Phe Giu Giu Ile Giu Leu Asp Asp Phe Ser Giu 355 360 365 Thr His Arg Leu Leu Pro Gin Asn Thr Ser Ser Thr Pro Val Gly Ser 370 375 380 Giy Val Arg Arg Ser Leu Ile Pro Thr Arg Giu Phe Ser Ala Thr Arg 385 390 395 400 Pro Thr Gly Val Vai Thr Tyr Giy Ser Pro Asp Thr Tyr Ser Ala Ser 405 410 415 Pro Val Thr Asp Pro Asp Ser Thr Ser Pro Ser Leu Val Ile Asp Asp 420 425 430 Thr Thr Thr Thr Pro Ile Ile Ile Ile Asp Gly His Thr Val Asp Leu 435 440 445 Tyr Ser Ser Asn Tyr Thr Leu His Pro Ser Leu Leu Arg Lys Arg Lys 450 455 460 Lys Arg Lys His Ala 465 SUBSTITUTE SHEET (RULE 26) WO 99/02694 i <210> 7 <211> 1410 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1)..(1410) <220> <223> Description of Artificial Sequence: Bovine papillomavirus type 1 L2 open reading frame (humanized) <220> <223> wild-type codons replaced with synonymous codons PCT/AU98/00530 used at relatively high frequency by human genes <400> 7 atg Met 1 agg Arg gtg Val1 gcc Al a gtg Val tcc Ser acc Thr ctt Leu gcc Ala agc gcc Ser Ala acc tgc Thr Cys gag ggc Glu. Gly atc tac Ile Tyr gcc gcc Ala Ala acc tcc Thr Ser cgc aag aga gtg aag cgc Arg aag Lys gac Asp ctg Leu ggc Gly tcc Ser tcC Ser 100 ctg Leu Lys 5 cag Gln acc Thr ggc Gly ggC Gly ctg Leu atc Ile cgc Arg gtg Val Arg Val Lys gcc ggc aca Ala Gly Thr atc gcC gac Ile Ala Asp 40 ggc ctg ggc Gly Leu Gly tca cca agg Ser Pro Arg 70 gcc tcc atc Ala Ser Ile ggc gcg ggC Gly Ala Gly cct ggc gtg Pro Gly Val 120 acc cct gac Thr Pro Asp 135 gcc Ala 10 cca Pro atc Ile gga Gly acc Thr tcc Ser 90 cct Pro gag Glu agc Ser cca Pro ctg Leu aca Thr cca Pro 75 aga Arg ctg Leu gac Asp tac gac ctg Tyr Asp Leu gtg atc- cga Val Ile Arg ttc ggc ggc Phe Gly Gly tct acc ggc Ser Thr Gly cgc acc gcc Arg Thr Ala gtg acc gcc Val Thr Ala acc ctg gaa Thr Leu Glu 110 gtg ctg ccc Val Leu Pro 125 tac Tyr aag Lys ctg Leu.

agg Arg ggC Gly ggg Gly act Thr gaa Glu cgc Arg ggg Gly cct gcc atc Pro Ala Ile gcc gtg Ala Val cct gca gac tcc ggc ctg Pro Ala Asp Ser Gly Leu 140 SUBSTITUTE SHEET (RULE 26) WO 99/02694 WO 9902694PCT/AU98/00530 gac Asp 145 ctg Leu ccc Pro tct Ser tct Ser ggc Gly 225 ac c Thr agc Ser tcC Ser ctg Leu gat Asp 305 gtg Val tac Tyr gag Glu gcc- Ala Ctg Leu ctg Leu gcc Ala ggt Gly 210 ggc Gly ccc Pro aag Lys tCC Ser aag Lys 290 acc Thr agg Arg acc Thr gag Giu ctg Leu gag Giu gac Asp tac Tyr 195 tta Leu gag Giu cgc Arg cgg Arg cag Gin 275 ggC Gly ctg Leu tac Tyr gtg Val cag Gin 355 tcc atc ggc Ser Ile Gly 150 aca gac tcc tcc Thr Asp Ser Ser cct Pro cgc Arg 180 cac His gaa Giu aac Asn tcC Ser tac Tyr 260 acc Thr cct Pro acc Thr tcc Ser gat Asp 340 gcc Aila gag Giu 165 cca Pro gcc Al a aat Asn atc Ile atc Ile 245 tac Tyr ttc Phe agc Ser aca Thr ctg Leu 325 gag Giu Gly ggc Giy ac c Thr cct Pro att Ile gag Giu 230 gcc Aia acc Thr gcc Aia ggc Gly cgt Arg 310 tcc Ser aac Asn ttc Phe ccc Pro tgg Trp ctc Leu ttt Phe 215 ctg Leu tcC Ser cag Gin aac Asn cgc Arg 295 agc Ser acc Thr acc Thr gag Giu gaa Glu c ag Gin c ag Gin 200 gta Val1 acc Thr aag Lys gtg Val Ccc Pro 280 gtg Val ggc Gly atc Ile cag Gin gag Giu 360 gac Asp gtg Val 185 ctg Leu gga Giy tac Tyr tcc Ser

CCC

Pro 265 ctg Leu ggc Gly aca Thr cat His ggc Gly 345 at c Ile ata Ile 170 agc Ser caa Gin ggc Gly ttC Phe cgc Arg 250 ac c Thr tac Tyr ctg Leu gag Giu gag Giu 330 ctg Leu gag Giu acc Thr 155 gcc Al a aat Asn tcc Ser tcg Ser ggc Giy 235 ggc Giy gaa Glu gag Giu tcc Ser gtg Vai 315 gat Asp gcc Al a ctc: Leu gag Giu gtg Vai gct Aia tcc Ser ggt Gly 220 tcC Ser atc Ile gat Asp gcc Al a cag Gin 300 ggc Giy gtg Vai ttc Phe gac Asp acc Thr ctg Leu gtg Val1 atc Ile 205 tta Leu ccc Pro ctg Leu ccc Pro gag Glu 285 gtg Vai ccc Pro gag Glu gtg Val gat Asp 365 ctg Leu gaa Giu cac His 190 gcc Ala ggg Gly cgc Arg aac Asn gaa Giu 270

CCC

Pro tac Tyr cag Gin gct Ala

CCC

Pro 350 ttc Phe atc Ile ctc Leu 175 cag Gin gag Giu gat Asp acc Thr tgg Trp 255 gtg Val gcc Ala aag Lys ctg Leu atc Ile 335 Ctg Leu agc Ser acc Thr 160 cag Gin tcc Ser aca Thr acc Thr agc Ser 240 ttc Phe ttc Phe gtg Val1 cct Pro cat His 320 ccc Pro cat His gag Glu 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 xvi acc cat cgc ctg ctg ccc cag aac ace tce tcc acc ccc gtg ggc age 1152 Thr His Arg Leu Leu Pro Gin Asn Thr Ser Ser Thr Pro Val Gly Ser 370 375 380 ggc gtg cgc aga age ctg atc cct acc cga gag ttc age gcc acc cgg 1200 Gly Val Arg Arg Ser Leu Ile Pro Thr Arg Glu Phe Ser Ala Thr Arg 385 390 395 400 cct acc ggc gtg gtg acc tac ggc tcc ccc gac acc tac tcc get age 1248 Pro Thr Gly Val Val Thr Tyr Gly Ser Pro Asp Thr Tyr Ser Ala Ser 405 410 415 ccc gtg acc gac cct gat tct acc tct cct age ctg gtg atc gac gac 1296 Pro Val Thr Asp Pro Asp Ser Thr Ser Pro Ser Leu Val Ile Asp Asp 420 425 430 acc acc acc acc ccc ate atc ate ate gac ggc cac aca gtg gat ctg 1344 Thr Thr Thr Thr Pro Ile Ile Ile Ile Asp Gly His Thr Val Asp Leu 435 440 445 tac agc age aac tac acc ctg cat ccc tcc ctg ctg agg aag cgc aag 1392 Tyr Ser Ser Asn Tyr Thr Leu His Pro Ser Leu Leu Arg Lys Arg Lys 450 455 460 aag cgc aag cat gcc taa 1410 Lys Arg Lys His Ala 465 470 <210> 8 <211> 469 <212> PRT <213> Artificial Sequence <400> 8 Met Ser Ala Arg Lys Arg Val Lys Arg Ala Ser Ala Tyr Asp Leu Tyr 1 5 10 Arg Thr Cys Lys Gin Ala Gly Thr Cys Pro Pro Asp Val Ile Arg Lys 25 Val Glu Gly Asp Thr Ile Ala Asp Lys Ile Leu Lys Phe Gly Gly Leu 40 Ala Ile Tyr Leu Gly Gly Leu Gly Ile Gly Thr Trp Ser Thr Gly Arg 55 Val Ala Ala Gly Gly Ser Pro Arg Tyr Thr Pro Leu Arg Thr Ala Gly 70 75 Ser Thr Ser Ser Leu Ala Ser Ile Gly Ser Arg Ala Val Thr Ala Gly 90 Thr Arg Pro Ser Ile Gly Ala Gly Ile Pro Leu Asp Thr Leu Glu Thr 100 105 110 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 xvii Leu Gly Ala Leu Arg Pro Gly Val Tyr Glu Asp Thr Val Leu Pro Glu 115 120 125 Ala Pro Ala Ile Val Thr Pro Asp Ala Val Pro Ala Asp Ser Gly Leu 130 135 140 Asp Ala Leu Ser Ile Gly Thr Asp Ser Ser Thr Glu Thr Leu Ile Thr 145 150 155 160 Leu Leu Glu Pro Glu Gly Pro Glu Asp Ile Ala Val Leu Glu Leu Gln 165 170 175 Pro Leu Asp Arg Pro Thr Trp Gin Val Ser Asn Ala Val His Gin Ser 180 185 190 Ser Ala Tyr His Ala Pro Leu Gin Leu Gin Ser Ser Ile Ala Glu Thr 195 200 205 Ser Gly Leu Glu Asn Ile Phe Val Gly Gly Ser Gly Leu Gly Asp Thr 210 215 220 Gly Gly Glu Asn Ile Glu Leu Thr Tyr Phe Gly Ser Pro Arg Thr Ser 225 230 235 240 Thr Pro Arg Ser Ile Ala Ser Lys Ser Arg Gly Ile Leu Asn Trp Phe 245 250 255 Ser Lys Arg Tyr Tyr Thr Gin Val Pro Thr Glu Asp Pro Glu Val Phe 260 265 270 Ser Ser Gin Thr Phe Ala Asn Pro Leu Tyr Glu Ala Glu Pro Ala Val 275 280 285 Leu Lys Gly Pro Ser Gly Arg Val Gly Leu Ser Gin Val Tyr Lys Pro 290 295 300 Asp Thr Leu Thr Thr Arg Ser Gly Thr Glu Val Gly Pro Gin Leu His 305 310 315 320 Val Arg Tyr Ser Leu Ser Thr Ile His Glu Asp Val Glu Ala Ile Pro 325 330 335 Tyr Thr Val Asp Glu Asn Thr Gin Gly Leu Ala Phe Val Pro Leu His 340 345 350 Glu Glu Gin Ala Gly Phe Glu Glu Ile Glu Leu Asp Asp Phe Ser Glu 355 360 365 Thr His Arg Leu Leu Pro Gin Asn Thr Ser Ser Thr Pro Val Gly Ser 370 375 380 Gly Val Arg Arg Ser Leu Ile Pro Thr Arg Glu Phe Ser Ala Thr Arg 385 390 395 400 Pro Thr Gly Val Val Thr Tyr Gly Ser Pro Asp Thr Tyr Ser Ala Ser 405 410 415 SUBSTITUTE SHEET (RULE 26) lfr k f.1% L'K A PCT/A iR8/00530 wVu YYvwuo4 xviii Pro Val Thr Asp Pro Asp Ser Thr Ser Pro Ser Leu Val Ile Asp Asp 420 425 430 Thr Thr Thr Thr Pro Ile Ile Ile Ile Asp Gly His Thr Val Asp Leu 435 440 445 Tyr Ser Ser Asn Tyr Thr Leu His Pro Ser Leu Leu Arg Lys Arg Lys 450 455 460 Lys Arg Lys His Ala 465 <210> 9 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Aequorea victoria gfp gene (humanized) <220> <221> CDS <222> <400> 9 atg age aag ggc gag gaa ctg ttc act ggc gtg gtc cca att ctc gtg 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 gaa ctg gat ggc gat gtg aat ggg cac aaa ttt tct gtc age gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 25 ggt gaa ggt gat gcc aca tac gga aag ctc acc ctg aaa ttc atc tgc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 acc act gga aag ctc cct gtg cca tgg cca aca ctg gtc act acc ttc 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 55 tct tat ggc gtg cag tgc ttt tcc aga tac cca gac cat atg aag cag 240 Ser Tyr Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gin 70 75 cat gac ttt ttc aag agc gcc atg ccc gag ggc tat gtg cag gag aga 288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg 90 acc ate ttt ttc aaa gat gac ggg aac tac aag ace cgc gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttc gaa ggt gac ace ctg gtg aat aga ate gag ctg aag ggc att 384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile v v SUBSTITUTE SHEET (RULE 26) W) 99/n3264 PCT/ARU98/00530 xix 115 120 125 gac ttt aag gag gat gga aac att ctc ggc cac aag ctg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tcc cac aat gtg tac ate atg gcc gac aag caa aag aat" ggc 480 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gin Lys Asn Gly 145 150 155 160 ate aag gtc aac ttc aag atc aga cac aac att gag gat gga tcc gtg 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 cag ctg gcc gac cat tat caa cag aac act cca ate ggc gac ggc cct 576 Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtg ctc ctc cca gac aac cat tac ctg tcc acc cag tct gcc ctg tct 624 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc ctg ctg gag ttt gtg 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 acc gct get ggg ate aca cat ggc atg gac gag ctg tac aag tga 717 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> <211> 238 <212> PRT <213> Artificial Sequence <400> Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 25 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 55 Ser Tyr Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gin 70 75 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg 90 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 Lys PI Asp PI 1: Tyr A 145 Ile L} Gin L Val L Lys A! 2: Thr A: 225 he he sn ys eu eu sp 10 la Glu 115 Lys Ser Val Ala Leu 195 Pro Ala Gly Glu His Asn Asp 180 Pro Asn Gly Thr Gly Val 150 Lys Tyr Asn Lys Thr 230 Leu Asn 135 Tyr Ile Gin His Arg 215 His Val 120 Ile Ile Arg.

Gin Tyr 200 Asp Gly Arg Gly Ala Asn 170 Thr Ser Met Asp Ile His Asp 155 Ile Pro Thr Val Glu 235 Glu Lys 140 Lys Glu Ile Gin Leu 220 Leu Leu 125 Leu Gin Asp Gly Ser 205 Leu Tyr Lys Glu Lys Gly Asp 190 Ala Glu Lys Gly Tyr Asn Ser 175 Gly Leu Phe Ile Asn Gly 160 Val Pro Ser Val <210> 11 <211> 717 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> <220> <223> Description of Artificial Sequence: Synthetic gfp gene (Papillomavirusized) <220> <223> Codons of humanized gfp gene replaced with synonymous codons used at relatively high frequency by papillomavirus genes <400> 11 atg agt aaa ggg gaa gaa cta ttt aca ggg gtg gtg cct ata Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 1 5 10 S gaa cta gat ggg gat gtg aat ggg cac aaa ttt tct gtc agt Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 25 ggg gaa ggg gat gca aca tat ggg aaa cta aca cta aaa ttt Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 40 cta gtg Leu Val ggg gaa Gly Glu ata tgc Ile Cys 48 96 144 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 aca aca Thr Thr ggg aaa cta cct gtg cca tgg cct aca Gly Lys Leu Pro Val Pro Trp Pro Thr gtg aca aca ttt Val Thr Thr Phe agt Ser tat ggg gtg caa Tyr Gly Val Gin tgc Cys 70 ttt agt aga tat Phe Ser Arg Tyr cct Pro gat cat atg aaa Asp His Met Lys 240 cat gat ttt ttt His Asp Phe Phe agt gca atg ccc gag ggg tat gtg caa Ser Ala Met Pro Glu Gly Tyr Val Gin gaa aga Glu Arg 288 aca ata ttt Thr Ile Phe aaa ttt gaa Lys Phe Glu 115 aaa gat gat ggg Lys Asp Asp Gly aat Asn 105 tat aaa aca aga Tyr Lys Thr Arg gca gaa gtc Ala Glu Val 110 aaa ggg ata Lys Gly Ile ggg gat aca cta Gly Asp Thr Leu aat aga ata gag Asn Arg Ile Glu gat ttt- Asp Phe 130 aaa gaa gat ggg Lys Glu Asp Gly aat Asn 135 ata cta ggg cat Ile Leu Gly His cta gaa tat aat Leu Glu Tyr Asn tat Tyr 145 aat agt cat aat Asn Ser His Asn gtg Val 150 tat ata atg gca Tyr Ile Met Ala gat Asp 155 aaa caa aaa aat Lys Gin Lys Asn ggg Gly 160 ata aaa gtg aat Ile Lys Val Asn aaa ata ata aga Lys Ile Ile Arg cat His 170 ata gaa gat gga Ile Glu Asp Gly tec gtg Ser Val 175 caa cta gca Gin Leu Ala gtg cta cta Val Leu Leu 195 gat Asp 180 cat tat caa caa His Tyr Gin Gin aat Asn 185 aca cct ata ggg Thr Pro Ile Gly gat ggg cct Asp Gly Pro 190 gca cta agt Ala Leu Ser cct gat aac cat Pro Asp Asn His tat Tyr 200 cta agt aca caa Leu Ser Thr Gin agt Ser 205 aaa gat Lys Asp 210 cct aat gaa aaa Pro Asn Glu Lys aga Arg 215 gat cat atg gtg Asp His Met Val cta Leu 220 ctc gag ttt gtg Leu Glu Phe Val 672 717 aca Thr 225 gca gca ggg ata Ala Ala Gly Ile aca Thr 230 cat ggg atg gat His Gly Met Asp gaa Glu 235 cta tat aaa tga Leu Tyr Lys <210> 12 <211> 238 <212> PRT <213> Artificial Sequence <400> 12 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530 xxii Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 25 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 40 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 55 Ser Tyr Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gin 70 75 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg 90 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gin Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Ile Arg His Ile Glu Asp Gly Ser Val 165 170 175 Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Ala(GCA) <400> 13 taaggactgt aagactt 17 SUBSTITUTE SHEET (RULE 26) WO 99/n2 d PCT/AU98/00530 xxiii <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific- for-Arg-(CGA) <400> 14 cgagccagcc aggagtc 17 <210> <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Asn(AAC) <400> ctagattggc-aggaatt 17 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Asp(GAC) <400> 16 taagatatat agattat 17 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Cys(TGC) <400> 17 aagtcttagt agagatt 17 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence VWVV VV SUBSTITUTE SHEET (RULE 26) wn oo/')Ao PCT/A U98/00530 xxiv <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Glu(GAA) <400> 18 tatttctaca cagcatt 17 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Gln(CAA) <400> 19 ctaggacaat aggaatt 17 <210> <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Gly(GGA) <400> tactctcttc tgggttt 17 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for His(CAC) <400> 21 tgccgtgact cggattc 17 <210> 22 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Ile(ATC) <400> 22 SUBSTITUTE SHEET (RULE 26) WO 99/02694 PCT/AU98/00530

XXV

tagaaataag agggctt 17 <210> 23 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Leu(CTA) <400> 23 tacttttatt tggattt 17 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Leu(CTT) <400> 24 tattagggag aggattt 17 <210> <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Lys(AAA) <400> tcactatgga gatttta 17 <210> 26 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Lys(AAG) <400> 26 cgcccaacgt ggggctc 17 <210> 27 <211> 17 SUBSTITUTE SHEET (RULE 26) 114 69 nQnIfOA PCT/AU98 /n0530 f 7 laP xxvi <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Met (elong) <400> 27 tagtacggga aggattt 17 S <210> 28 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Phe(TTC) <400> 28 tgtttatggg atacaat 17 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Pro(CCA) <400> 29 tcaagaagaa ggagcta 17 <210> <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Pro(CCI) <400> gggctcgtcc gggattt 17 <210> 31 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SUBSTITUTE SHEET (RULE 26) wO QQ/06Qd PCT/AU98/00530 xxvii Oligonucleotide specific for Ser(AGC) <400> 31 ataagaaagg aagatcg 17 <210> 32 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Thr(ACA) <400> 32 tgtcttgaga agagaag 17 <210> 33 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Tyr(TAC) <400> 33 tggtaaaaag aggattt 17 <210> 34 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide specific for Val(GTA) <400> 34 tcagagtgtt cattggt 17 t: SUBSTITUTE SHEET (RULE 26)

Claims

1. A method of constructing a synthetic polynucleotide from which a protein is selectively expressible in a target cell of a mammal, relative to another cell of the mammal, said method comprising: selecting a first codon of a parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a higher translational efficiency in said target cell than in said other cell; and replacing said first codon with said synonymous codon to form said synthetic polynucleotide.

2. The method of claim 1, wherein said first codon and said synonymous codon are selected by: 9 comparing translational efficiencies of individual codons in said target cell relative to said other cell; and selecting said first codon and said synonymous codon based on said comparison.

3. The method of claim 2, wherein the translational 9 .efficiency of an individual codon is compared by 25 measuring the abundance of an iso--tRNA corresponding to said individual codon in said target cell relative to said other cell.

4. The method of claim 3, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said target cell relative to said other cell. The method of claim 3, wherein selecting said first codon and said synonymous codon comprises: measuring abundance of different iso-tRNAs in said target cell relative to said other cell; and selecting said first codon and said synonymous codon based on said measurement, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said target cell than in said other cell.

6. The method of claim 3, wherein said synonymous codon corresponds to an iso-tRNA that is present in said target cell at a level which is at least 110% of 15 the level of the iso-tRNA that is present in said other Scell.

7. The method of claim 1, wherein said synonymous codon is selected from the group consisting of a codon used at relatively high frequency by genes of said target cell, a codon used at relatively high frequency by genes of the mammal, a codon used at relatively low frequency by genes of said other cell, and a codon used at relatively low frequency by S 25 genes of an organism other than said mammal.

8. The method of claim 1, wherein said first codon and said synonymous codon are selected such that said protein is expressed from said synthetic polynucleotide in said target cell at a level which is at least 110% of the level at which said protein is expressed from said parent polynucleotide in said target cell.

9. The method of claim 1, wherein said other cell is a precursor cell of said target cell.

10. The method of claim 1, wherein said other cell is a cell derived from said target cell.

11. The method of claim 1, wherein said target cell is of the same type as the other cell, but at a different stage of differentiation.

12. The method of claim 1, wherein said target cell is of the same type as the other cell, but at a different stage of the cell cycle. S.

13. A synthetic polynucleotide constructed according to the method of any one of claims 1 to 12.

14. A vector comprising the synthetic polynucleotide of claim 13 operably linked to one or more regulatory .i nucleotide sequences.

15. A pharmaceutical composition comprising the synthetic polynucleotide of claims 13, together with a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising the vector of claim 14, together with a pharmaceutically acceptable carrier.

17. A cell comprising the synthetic polynucleotide of claim 13.

18. A cell comprising the vector of claim 14.

19. A synthetic polynucleotide from which a protein is produced at a higher level in a target cell of a mammal than in another cell of the mammal, wherein a first codon in a parent polynucleotide encoding said protein has been replaced with a synonymous codon, wherein said synonymous codon has been selected on the basis that exhibits a higher translational efficiency in said target cell than in said other cell, wherein if said synonymous codon is independently selected from the group consisting of gcc, cgc, aac, gac, tgc, cag, ggc, cac, atc, ctg, aag, ccc, ttc, agc, acc, tac, gtg, ggg, 15 att, etc, tcc, and gtc, then at least one other codon in said parent polynucleotide has been replaced with another synonymous codon that is not selected from said group. *e

20. A synthetic polynucleotide from which a protein is produced at a higher level in a differentiated cell of a mammal than in an undifferentiated cell of the mammal, wherein a first codon in a parent polynucleotide encoding said protein has been replaced with a synonymous codon, wherein said synonymous codon has been selected on the basis that exhibits a higher translational efficiency in said differentiated cell than in said undifferentiated cell, each said synonymous codon being independently selected from the group consisting of gca (Ala), cuu (Leu) and cua (Leu).

21. The synthetic polynucleotide of claim 20, wherein said differentiated cell is a differentiated keratinocyte.

22. A synthetic polynucleotide from which a protein is produced at a higher level in an undifferentiated cell of a mammal than in a differentiated cell of the mammal, wherein a first codon in a parent polynucleotide encoding said protein has been replaced with a synonymous codon, wherein said synonymous codon has been selected on the basis that exhibits a higher translational efficiency in said undifferentiated cell than in said differentiated cell, wherein each said synonymous codon is independently selected from the 15 group consisting of cga (Arg) cci (Pro) and aac (Asn) and wherein if said synonymous codon is aac (Asn) then at least one other codon in the parent polynucleotide has been replaced with another 9. synonymous codon that is selected from the group consisting of cga (Arg) and cci (Pro).

23. The synthetic polynucleotide of claim 22, wherein said undifferentiated cell is an undifferentiated 9 keratinocyte.

24. A method of selectively expressing a protein in a 9 target cell of a mammal, said method comprising: selecting a first codon of a parent polynucleotide encoding said protein for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a higher translational efficiency in said target cell than in another cell of said mammal; -replacing said first codon with said synonymous codon to construct a synthetic polynucleotide; and introducing said synthetic polynucleotide into a cell selected from the group consisting of said target cell and a precursor of said target cell, said synthetic polynucleotide being operably linked to a regulatory polynucleotide, whereby said protein is selectively expressed in said target cell. The method of claim 24, wherein said synonymous codon has a higher translational efficiency in said 15 target cell than in said other cell. 4

26. A method of expressing a protein from a first polynucleotide in a target cell, said method *comprising: introducing into said target cell a second polynucleotide encoding an iso-tRNA, wherein said fo second polynucleotide is operably linked to one or more regulatory nucleotide sequences, and wherein said iso-tRNA is normally in relatively low abundance in comparison to other iso-tRNAs in said target cell *4 and corresponds to a codon of said first a polynucleotide; and expressing said second polynucleotide in said target cell, whereby said protein is expressed in said target cell. 79

27. A method of expressing a protein from a first polynucleotide in a target cell, said method comprising introducing into a precursor of said target cell a second polynucleotide encoding an iso-tRNA, wherein said second polynucleotide is operably linked to one or more regulatory nucleotide sequences, and wherein said iso-tRNA is normally in relatively low abundance in said target cell and corresponds to a codon of said first polynucleotide, wherein said precursor is exposed to conditions sufficient to produce said target cell; and expressing said second polynucleotide in said target cell, whereby said protein is expressed from said first polynucleotide in said target cell.

28. A method of producing a virus particle in a cycling eukaryotic cell, wherein said virus particle comprises a protein necessary for assembly of said virus particle, and wherein said protein is expressed 20 in said cell from a parent polynucleotide, but not at a level sufficient to permit virus assembly therein, said method comprising: replacing a first codon of said parent polynucleotide with a synonymous codon to produce a S 25 synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide, such that said protein is expressible from said synthetic polynucleotide in said cell at a level sufficient to permit virus assembly therein; and introducing into said cell said synthetic polynucleotide operably linked to one or more regulatory nucleotide sequences, wherein said cycling eukaryotic cell comprises said protein necessary for assembly of said virus particle, whereby said protein is expressed and said virus particle is produced in said cell.

29. The method of claim 28, wherein said synonymous codon has a higher translational efficiency in said cell than said first codon.

30. A method of producing a virus particle in a cycling cell, wherein said virus particle comprises at least one protein necessary for assembly of said virus particle, wherein said protein is expressed in said cell from a first polynucleotide, but not at a level 15 sufficient to permit virus assembly therein, and wherein the abundance of an iso-tRNA specific for a codon of said first polynucleotide limits the rate of production of said protein, said method comprising: introducing into said cell a second polynucleotide 20 encoding said iso-tRNA; and expressing said second polynucleotide in said cell, whereby said virus particle is produced in said cell. *o 25 31. A method of producing a virus particle in a cycling eukaryotic cell, wherein said virus particle comprises a protein necessary for assembly of said virus particle, and wherein said protein is expressed in said cell from a parent polynucleotide, but not at a level sufficient to permit virus assembly therein, said method comprising: 81 replacing a first codon of said parent polynucleotide with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide, such that said protein is expressible from said synthetic polynucleotide in said cell at a level sufficient to permit virus assembly therein; introducing into a precursor of said cell said synthetic polynucleotide operably linked to one or more regulatory nucleotide sequences; and exposing said precursor to conditions sufficient to produce said cell, whereby said protein is expressed and said virus particle is produced in said cell.

32. A vector comprising the synthetic polynucleotide of any one of claims 19 to 23, wherein said synthetic polynucleotide is operably linked to one or more regulatory nucleotide sequences.

33. A pharmaceutical composition comprising the synthetic polynucleotide of any one of claims 19 to 23, together with a pharmaceutically acceptable carrier. 25 34. A pharmaceutical composition comprising the vector of claim 32, together with a pharmaceutically acceptable carrier. A cell comprising the synthetic polynucleotide of any one of claims 19 to 23.

36. A cell comprising the vector of claim 32.

37. A cell produced by the method of claim 24 or claim

38. A cell produced by the method of claim 20 or claim 21.

39. A virus particle produced by the method of claim 28 or claim 29. A virus particle produced by the method of claim .30 or claim 31.

41. A method of constructing a synthetic 15 polynucleotide from which a protein is expressible at a higher level in a first cell of a mammal than in a second cell of the mammal, said method comprising: selecting a first codon of a parent polynucleotide for replacement with a synonymous codon, wherein said 20 synonymous codon is selected on the basis that it exhibits a higher translational efficiency in said first cell than in said second cell; and replacing said first codon with said synonymous codon to form said synthetic polynucleotide.

42. The method of claim 41, wherein said first codon and said synonymous codon are selected by: comparing translational efficiencies of individual codons in said first cell relative to said second cell; and selecting said first codon and said synonymous codon based on said comparison.

43. The method of claim 42, wherein the translational efficiency of an individual codon is compared by measuring the abundance of an iso-tRNA corresponding to said individual codon in said first cell relative to said second cell.

44. The method of claim 43, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said first cell relative to said second cell.

45. The method of claim 43, wherein selecting said first codon and said synonymous codon comprises: 15 measuring abundance of different iso-tRNAs in said first cell relative to said second cell; and selecting said first codon and said synonymous codon based on said measurement, wherein said synonymous codon corresponds to an iso-tRNA which is in higher 20 abundance in said first cell than in said second cell.

46. The method of claim 43, wherein said synonymous codon corresponds to an iso-tRNA that is present in said first cell at a level which is at least 110% of 25 the level of the iso-tRNA that is present in said second cell.

47. The method of claim 41, wherein said synonymous codon is selected from the group consisting of a codon used at relatively high frequency by genes of said first cell, a codon used at relatively high frequency by genes of the mammal, a codon used at 84 relatively low frequency by genes of said second cell, and a codon used at relatively low frequency by genes of an organism other than said mammal.

48. The method of claim 41, wherein said first codon and said synonymous codon are selected such that said protein is expressed from said synthetic polynucleotide in said first cell at a level which is at least 110% of the level at which said protein is expressed from said parent polynucleotide in said first cell.

49. The method of claim 41, wherein said other cell is a precursor cell of said second cell. 15 50. The method of claim 41, wherein said other cell is a cell derived from said second cell.

51. A method of constructing a synthetic polynucleotide from which a protein is expressible at a 20 lower level in a first cell of a mammal than in a second cell of the mammal, said method comprising: selecting a first codon of a parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it 25 exhibits a lower translational efficiency in said first cell than in said second cell; and replacing said first codon with said synonymous codon to form said synthetic polynucleotide.

52. The method of claim 51, wherein said first codon and said synonymous codon are selected by: Scomparing translational efficiencies of individual codons in said first cell relative to said second cell; and selecting said first codon and said synonymous codon based on said comparison.

53. The method of claim 52, wherein the translational efficiency of an individual codon is compared by measuring the abundance of an iso-tRNA corresponding to said individual codon in said first cell relative to said second cell.

54. The method of claim 53, wherein said synonymous codon corresponds to an iso-tRNA which is in lower 15 abundance in said first cell relative to said second cell. The method of claim 53, wherein selecting said first codon and said synonymous codon comprises: measuring abundance of different iso-tRNAs in said first cell relative to said second cell; and selecting said first codon and said synonymous codon based on said measurement, wherein said .synonymous codon corresponds to an iso-tRNA which is 25 in lower abundance in said first cell than in said second cell.

56. A method of constructing a synthetic polynucleotide from which a protein is expressible at a higher level than from a parent polynucleotide in a cell of a mammal, said method comprising: providing a comparison of translational efficiencies of individual codons in said cell; selecting from said comparison a first codon of said parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a higher translational efficiency in said cell than said first codon; and replacing said first codon with said synonymous codon to construct said synthetic polynucleotide.

57. The method of claim 56, wherein said synonymous codon corresponds to an iso-tRNA which is in higher abundance in said cell type relative to the iso-tRNA corresponding to said first codon.

58. The method of claim 56, wherein said first codon and said synonymous codon are selected such that said protein is expressed from said synthetic polynucleotide S. 20 in said cell type at a level which is at least 110% of the level at which said protein is expressed from said parent polynucleotide in said cell type.

59. The method of claim 56, wherein said comparison is 25 provided by comparing translational efficiencies of individual codons in said cell type. The method of claim 57, wherein said translational efficiencies are compared by measuring abundance of different iso-tRNAs in said cell type. 87

61. A method of constructing a synthetic polynucleotide from which a protein is expressible at a lower level than from a parent polynucleotide in a cell of a mammal, said method comprising: providing a comparison of translational efficiencies of individual codons in said cell; selecting from said comparison a first codon of said parent polynucleotide for replacement with a synonymous codon, wherein said synonymous codon is selected on the basis that it exhibits a lower translational efficiency in said cell than said first codon; and replacing said first codon with said synonymous Scodon to construct said synthetic polynucleotide.

62. The method of claim 61, wherein said synonymous S: codon corresponds to an iso-tRNA which is in lower abundance in said cell type relative to the iso-tRNA corresponding to said first codon.

63. The method of claim 61, wherein said comparison is provided by comparing translational efficiencies of individual codons in said cell type. oo S 25 64. The method of claim 61, wherein said translational efficiencies are compared by measuring abundance of different iso-tRNAs in said cell type. A synthetic polynucleotide constructed according to the method of any one of claims 41 to 64. 88

66. A vector comprising the synthetic polynucleotide of claim 65 operably linked to one or more regulatory nucleotide sequences.

67. A pharmaceutical composition comprising the synthetic polynucleotide of claims 65, together with a pharmaceutically acceptable carrier.

68. A pharmaceutical composition comprising the vector of claim 66, together with a pharmaceutically acceptable carrier.

69. A cell comprising the synthetic polynucleotide of S" claim A cell comprising the vector of claim 66.

71. A method of expressing a protein in a cell of a S. mammal, said method comprising: introducing into said cell a vector comprising a synthetic polynucleotide constructed according to the method of any one of claims 56 to 60, wherein said O synthetic polynucleotide is operably linked to one or lo more regulatory nucleotide sequences; and 25 expressing said synthetic polynucleotide in said cell, whereby said protein is expressed from said synthetic polynucleotide in said cell.

72. The method of any one of claims 1 to 12, or the polynucleotide of claim 13, or the vector of claim 14, or the pharmaceutical composition of claim 15 or claim 16, or the cell of claim 17 or claim 18, or the polynucleotide of any one of claims 19 to 23, or the method of any one of claims 24 to 31, or the vector of claim 32, or the composition of claim 33 or claim 34, or the cell of any one claim 35 to 38, or the virus particle of claim 39 or claim 40, or the method of any one of claims 41 to 64, or the polynucleotide of claim or the vector of claim 66, or the composition of claim 67 or claim 68, or the cell of claim 69 or claim or the method of claim 71, substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this Sixth Day of March, 2002 9** THE UNIVERSITY OF QUEENSLAND 15 DAVIES COLLISON CAVE Patent Attorneys for the Applicant 4 *e•