Patent Grant
7741079
While gene therapy is of great clinical interest for treatment of gene defects, this therapy has not entered into mainstream clinical practice, at least in part 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 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 (e.g., 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 SV40. 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.
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 control proteins have been unrewarding.
Blockage of 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-1 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. The present inventors have discovered the mechanism by which L1 protein is expressed in the late stage of the life cycle of this virus, and have also discovered a method of general application whereby polynucleotides can be designed or modified in order to effect selective expression of a protein in a target cell or tissue.
Accordingly, in one aspect of the invention, there is provided 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:
Preferably, said first codon and said synonymous codon are selected by:
A translational efficiency of a codon may be determined by any suitable technique. In a preferred embodiment, the translational efficiency of a codon is measured by:
Preferably, the above method is further characterized by:
Suitably, this method is further characterized by:
The above method may be further characterized by the step of introducing the synthetic construct into an organism or part thereof such that said synthetic construct is introduced into said target cell or said other cell.
Preferably, said synonymous codon corresponds to a reporter construct from which the reporter protein is expressed in said target cell at a level that is at least 110%, preferably at least 200%, more preferably at least 500%, and most preferably at least 1000%, of that expressed from the same reporter construct in said other cell.
In an alternate embodiment, the translational efficiency of a codon is compared 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 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:
Alternatively, the step of selecting may be characterized in that a synonymous codon according to the invention is selected from the group consisting of (1) a codon used at relatively high frequency by genes, preferably highly expressed genes, of a said target cell or tissue, (2) a codon used at relatively high frequency by genes, preferably highly expressed genes, of the mammal, (3) a codon used at relatively low frequency by genes of a said one or more other cells or tissues, and (4) a codon used at relatively low frequency by genes of another organism.
The step of selecting may be characterized in that a first codon according to the invention is selected from the group consisting of (a) a codon used at relatively high frequency by genes, preferably highly expressed genes, of a said one or more other cells or tissues, (b) a codon used at relatively low frequency by genes of a said target cell or tissue, (c) a codon used at relatively low frequency by genes of the mammal, and (d) a codon used at relatively high frequency by genes of another organism.
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.
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 differentiated 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).
Synonymous codon(s) are preferably selected from the group consisting of aga (Arg), cgg (Arg), tgc (Cys), gga (Gly), ggc (Gly), ccg (Pro), cga (Pro), aca (Thr), acg (Thr), and act (Thr), when said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
Suitably, the first codon is selected from the group consisting of agg (Arg), tgt (Cys), ggg (Gly), ggt (Gly), ccc (Pro), cct (Pro), and acc (Thr), when said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
In yet another aspect, the invention resides in a method for selectively expressing a protein in a target cell or tissue of a mammal, said method comprising:
In yet another aspect, the invention provides a method of expressing a protein in a target cell from a first polynucleotide, said method comprising:
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 sufficient to permit virus assembly therein, said method comprising:
In yet a further aspect of the invention, there is provided 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 not expressed in said cell from a parent polynucleotide at a level sufficient to permit virus assembly therein, and wherein a codon of said parent polynucleotide is rate limiting for the production of said protein, said method comprising introducing into said cell a polynucleotide capable of expressing therein an iso-tRNA specific for said codon. The virus particle is thereby produced in the cycling cell.
The invention also includes cells, vectors, and viruses made by any of the methods described above, and pharmaceutical compositions comprising one or more such cells, vectors, and viruses together with a pharmaceutically acceptable carrier.
This invention relates generally to gene therapy. More particularly, the present invention relates to a synthetic polynucleotide 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 polynucleotide encoding the protein has been replaced with a synonymous codon. The invention also relates to production of virus particles using one or more synthetic polynucleotides and the method according to the invention.
While investigating the mechanism by which protein expression is controlled during the life cycle of a bovine papillomavirus (PV), the inventors, surprisingly, discovered that PV L1 protein can be produced at 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 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 polynucleotides 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.
1. Definitions
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
By “expressible” is meant expression of a protein to a level sufficient to effect a particular function associated with the protein. By contrast, the terms “not expressible” and “not substantially expressible” as used interchangeably herein refers to (a) no expression of a protein, (b) expression of a protein to a level that is not sufficient to effect a particular function associated with the protein, (c) expression of a protein, which cannot be detected by a monoclonal antibody specific for the protein, or (d) expression of a protein, which is less that 1% of the level expressed in a wild-type cell that normally expresses the protein.
By “expressing said synthetic construct” is meant transcribing the synthetic construct such that mRNA is produced.
By “expression vector” is meant any autonomous genetic element capable of directing the synthesis of a protein encoded by the vector. Such expression vectors are known by practitioners in the art.
As used herein, the term “function” refers to a biological, enzymatic, or therapeutic function.
By “highly expressed genes” is meant genes that express high levels of mRNA, and preferably high level of protein, relative to other genes.
By “isoaccepting transfer RNA” or “iso-tRNA” is meant one or more transfer RNA molecules that differ in their anticodon nucleotide sequence but are specific for the same amino acid.
By “natural gene” is meant a gene that naturally encodes the protein. However, it is possible that the parent polynucleotide encodes a protein that is not naturally-occurring but has been engineered using recombinant techniques.
The term “non-cycling cell” as used herein refers to a cell that has withdrawn from the cell cycle and has entered the G0 state. In this state, it is 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. By contrast, the term “cycling cell” as used herein refers to a cell, which is in one of the above phases of the cell cycle.
By “obtained from” is meant that a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source of the host. For example, the extract can be obtained from a tissue or a biological fluid isolated directly from the host.
The term “oligonucleotide” as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term “oligonucleotide” typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.
By “operably linked” is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.
By “pharmaceutically-acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.
“Polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
The term “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.
By “primer” is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotide residues, although it can contain fewer nucleotide residues. Primers can be large polynucleotides, such as from about 200 nucleotide residues to several kilobases or more. Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By “substantially complementary”, it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. Preferably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotide residues can be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non-complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
“Probe” refers to a molecule that binds to a specific sequence or sub-sequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the “target polynucleotide”, through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly.
The terms “precursor cell or tissue” and “progenitor cell or tissue” as used herein refer to a cell or tissue that can gives rise to a particular cell or tissue in which protein expression is to be targeted or in which translational efficiency of a codon is to be determined.
By “recombinant polypeptide” is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
“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 immobilized polynucleotides and the labeled polynucleotide.
“Stringent conditions” refers to temperature and ionic conditions under which only polynucleotides having a high frequency of complementary bases will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization. Generally, stringent conditions are selected to be about 10 to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.
The term “synthetic polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature. For example, the synthetic polynucleotide can be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.
The term “synonymous codon” as used herein refers to a codon having a different nucleotide sequence than another codon but encoding the same amino acid as that other codon.
By “translational efficiency” is meant the efficiency of a cell's protein synthesis machinery to incorporate the amino acid encoded by a codon into a nascent polypeptide chain. This efficiency can be evidenced, for example, by the rate at which the cell is able to synthesize the polypeptide from an RNA template comprising the codon, or by the amount of the polypeptide synthesized from such a template.
By “vector” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptII gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
2. Selection of Synonymous Codons
The present invention arises from the unexpected discovery that the translational efficiencies of different codons varies between different cells or tissues, or alternatively between cells or tissues in different states of differentiation or between cells in different stages of the cell cycle. Such differences can be exploited together with codon composition of a gene to regulate and direct expression of a protein to a particular cell or cell type, including cells in a selected tissue. Alternatively, these differences can be exploited together with codon composition of a gene to regulate and direct expression of a protein to a cell or tissue in a selected state of differentiation or in a selected stage of the cell cycle. According to the present invention, this selective targeting is effected by replacing at least one existing codon (sometimes referred to as a “first codon”) of a parent polynucleotide encoding the protein with a synonymous codon (i.e. one which encodes the same amino acid residue as the first 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 origin at high levels in in vitro mammalian cell culture systems, the main thrust of that method being the harvesting of such proteins. In distinct contrast, the present invention utilizes substitution of one or more codons in a gene to target expression of the gene to particular cells or tissues with the ultimate aim of facilitating gene therapy as described herein.
The present method preferably includes the step of selecting the codons such that the synonymous codon has a higher translational efficiency in said target cell or tissue (“cell or tissue” is sometimes referred to herein as “cell/tissue”) relative to said one or more other cells or tissues. As used herein, expression of a protein in a tissue refers alternatively to expression of the protein within a cell of the tissue or production of the protein within a cell and export of the protein from the cell to, for example, the extracellular matrix of a tissue.
Methods for determining translational efficiencies of different codons in and between different cells or tissues are described in detail in Section 3. The translational efficiencies so determined can be used to identify which isocoding triplets are differentially translated between the different cells or tissues. In a typical scenario, there will be: (A) codons with higher translational efficiencies in a target cell/tissue relative to one or more other cells/tissues; (B) codons with higher translational efficiencies in the one or more other cells/tissues relative to the target cell/tissue; and (C) codons with about the same translational efficiencies in the target cell/tissue relative to the one or more other cells/tissues. Synonymous codons are selected such that they correspond to (A) codons. Preferably, a synonymous codon is selected such that it has the largest difference in translational efficiency in the target cell or tissue relative to the existing codon that it replaces. Existing codons in a parent polynucleotide are preferably selected such that they do not have the same translational bias as the synonymous codons with respect to the target cell/tissue and the one or more other cell/tissue (i.e., existing codons should preferably not correspond to (A) codons). However, existing codons can have similar translational efficiencies in each of the target cell/tissue and the one or more other cells/tissues (i.e., existing codons can correspond to (C) codons. They can also have a translational bias opposite to that of the synonymous codons (i.e., existing codons can, and preferably do, correspond to (B) codons).
Suitably, a synonymous codon has a translational efficiency in the target cell/tissue that is at least 110%, preferably at least 200%, more preferably at least 500%, and still more preferably at least 1000%, of that in the other cell(s)/tissue(s). In the case of two or more synonymous codons having similar translational efficiencies in the target cell/tissue relative to the other cell(s)/tissue(s), it will be appreciated that any one of these codons can be used to replace the existing codon.
It is preferable but not necessary to replace all the existing codons of the parent polynucleotide with synonymous codons having higher translational efficiencies in the target cell/tissue compared to the other cells/tissues. Increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of the parent polynucleotide.
The difference in level of protein expressed in the target cell/tissue from a synthetic polynucleotide relative to that expressed in the other cell(s)/tissue(s) depends on the percentage of existing codons replaced by synonymous codons, and the difference in translational efficiencies of the synonymous codons in the target cell/tissue relative to the other cell(s)/tissue(s). Put another way, the fewer such replacements, and/or the smaller the difference in translational efficiencies of the synonymous codons between the different cells/tissues, the smaller the difference in protein expression between the target cell/tissue and the other cell(s)/tissue(s) will be. Conversely, the more such replacements, and/or the greater the difference in translational efficiencies of the synonymous codons between the different cells/tissues, the greater the difference in protein expression between the target cell/tissue and the other cell(s)/tissue(s) will be. The inventors have found in this respect that a protein can be expressed from a synthetic polynucleotide in a target cell/tissue at levels greater than 10,000-fold over those expressed in another cell/tissue.
In contrast to differential protein expression between different cells/tissues, it will be appreciated that a synthetic polynucleotide may be tailored with synonymous codons such that expression of a protein in a target cell is enhanced. In this regard, the difference in level of protein expressed in the target cell/tissue from a synthetic polynucleotide relative to that expressed from a parent polynucleotide depends on the percentage of existing codons replaced by synonymous codons, and the difference in translational efficiencies between the existing codons and the synonymous codons in the target cell/tissue. Put another way, the fewer such replacements, and/or the smaller the difference in translational efficiencies between the synonymous and existing codons, the smaller the difference in protein expression between the synthetic polynucleotide and parent polynucleotide will be. Conversely, the more such replacements, and/or the greater the difference in translational efficiencies between the synonymous and existing codons, the greater the difference in protein expression between the synthetic polynucleotide and parent polynucleotide will be. The inventors have found in this respect that a protein can be expressed from a synthetic polynucleotide in a target cell/tissue at levels greater than 10,000-fold than from a parent polynucleotide.
Preferably, the at least one existing codon and the synonymous codon are selected 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.
In a preferred embodiment, the synonymous codon is a codon which has a higher translational efficiency in the target cell or tissue relative to a precursor cell or tissue of the target cell or tissue.
In an alternate embodiment, the synonymous codon is a codon which has a higher translational efficiency in the target cell or tissue relative to a cell or tissue derived from said target cell or tissue.
The two codons can be selected by measuring translational efficiencies of different codons in the target cell or tissue relative to the one or more other cells or tissues and identifying the at least one existing codon and the synonymous codon based on this measurement.
Synonymous codon(s) are preferably selected from the group consisting of aga (Arg), cgg (Arg), tgc (Cys), gga (Gly), ggc (Gly), ccg (Pro), cga (Pro), aca (Thr), acg (Thr), and act (Thr), wherein said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
Suitably, the at least one existing codon is selected from the group consisting of agg (Arg), tgt (Cys), ggg (Gly), ggt (Gly), ccc (Pro), cct (Pro), and acc (Thr), wherein said target cell is an undifferentiated cell, and preferably an undifferentiated epithelial cell.
Suitably, synonymous codons for higher level expression of a protein in a differentiated cell, preferably a differentiated keratinocyte, are selected from the group consisting of gca (Ala), cuu (Leu) and cua (Leu).
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).
3. Methods of Determining Codon Translational Efficiency
3.1. Expressing a Synthetic Construct Comprising a Tandem Repeat of Identical Codons Fused in Frame to a Reporter Polynucleotide
A major aspect of the present invention is based, at least in part, on the discovery that different but synonymous stretches of identical codons fused respectively in frame with a reporter polynucleotide can give rise to different levels of reporter protein expressed within a given cell type. Not wishing to be bound by any particular theory, it is believed that a tandem series of identical codons causes a ribosome to pause during translation if the iso-tRNA corresponding to the identical codons is limiting. In this regard, it is known that ribosomal pausing leads to a failure to complete a nascent polypeptide chain and an uncoupling of transcription and translation. Accordingly, the levels of reporter protein expressed in the different cells or tissues are sensitive to the intracellular abundance of the iso-tRNA species corresponding to the identical codons and therefore provide a direct correlation of a cell's or tissue's preference for translating a given codon. This means, for example, that if the levels of the reporter protein obtained in a cell or tissue type to which a synthetic construct having a tandem series of identical first codons is provided are lower than the levels expressed in the same cell or tissue type to which a different synthetic construct having a tandem series of identical second codons is provided (i.e. wherein the first codons are different from, but synonymous with, the second codons), then it can be deduced that the cell or tissue has a higher preference for the second codon relative to the first codon with respect to translation. Put another way, the second codon has a higher translational efficiency compared to the first codon in the cell or tissue type.
With regard to differential protein expression between different cell or tissue types, it will be appreciated that if the levels of the reporter protein obtained in a target cell or tissue type to which a synthetic construct having a tandem series of identical codons is provided are lower than the levels expressed in the another cell or tissue type to which the same synthetic construct is provided, then it can be deduced that the target cell or tissue has a higher preference for the codon relative to the other cell or tissue with respect to translation. Put another way, the codon has a higher translational efficiency in the target cell or tissue compared to the other cell or tissue type.
Suitably, the tandem repeat comprises at least three identical codons. Preferably, the tandem repeat comprises four identical codons, more preferably five or seven identical codons and most preferably six identical codons.
The tandem repeat can be fused at a location adjacent to, or within, the reporter polynucleotide. The location is preferably selected such that the tandem repeat interferes with translation of at least a detectable portion of the reporter protein such that expression of the protein can be detected or assessed. Preferably, the tandem repeat is located immediately upstream (translationally) from the reporter polynucleotide.
It is of course possible that a tandem repeat of identical amino acid residues (e.g., an oligo-proline repeat) can render the reporter protein unstable. Typically, protein instability is detected when expression of the reporter gene is not detectable with any choice of isoaccepting codon specific for the amino acid corresponding to the tandem repeat. The inventors have found in this regard that protein instability can be alleviated by use of at least one spacer codon within the tandem repeat of identical codons, wherein the spacer codon encodes a neutral amino acid.
The at least one spacer codon can be placed adjacent to, or interposed between, some or all of the identical codons corresponding to the tandem repeat. For example, a suitable interposition for a penta-repeat of identical codons can be selected from the group consisting of: (a) I-S-I-S-I-S-I-S-I-S; (b) S-I-S-I-S-I-S-I-S-I; (c) I-S-I-S-I-I-S-I; (d) I-S-I-I-S-I-S-I; (e) I-S-I-S-I-I-I; (f) I-I-S-I-S-I-I; (g) I-I-I-S-I-S-I; (h) I-S-I-I-S-I-I; (i) I-I-S-I-I-S-I; (j) I-S-I-I-I-S-I; (k) I-S-I-I-I-I; (l) I-I-S-I-I-I; (m) I-I-I-S-I-I; and (n) I-I-I-I-S-I, wherein I corresponds to an identical codon of a tandem repeat and S corresponds to a spacer codon.
Preferably, a spacer codon is efficiently translated in the cell or tissue type relative to other synonymous codons. This is important so that translation of the spacer codon is not rate limiting. The neutral amino acid includes, but is not restricted to, alanine and glycine.
The reporter polynucleotide can encode any suitable protein for which expression can be detected directly or indirectly such as by suitable assay. Suitable reporter polynucleotides include, but are not restricted to, polynucleotides encoding β-galactosidase, firefly luciferase, alkaline phosphatase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), herbicide resistance genes such as the bialophos resistance (BAR) gene that confers resistance to the herbicide BASTA, and green fluorescent protein (GFP). Assays for the activities associated with such proteins are known by those of skill in the art. Preferably, the reporter polynucleotide encodes GFP.
Persons of skill in the art will appreciate that reporter polynucleotides need not correspond to a full-length gene encoding a particular reporter protein. In this regard, the invention also contemplates reporter polynucleotide sub-sequences encoding desired portions of a parent reporter protein, wherein an activity or function of the parent protein is retained in said portions. A polynucleotide sub-sequence encodes a domain of the reporter protein having an activity associated therewith and preferably encodes at least 10, 20, 50, 100, 150, or 500 contiguous amino acid residues of the reporter protein.
The instant method is applicable to any suitable cell or tissue type and, hence, is not restricted to application to mammalian cells/tissues. Accordingly, the cell or tissue type can be of any animal or plant origin. The cell or tissue type can be of any suitable lineage. For example, a suitable cell can include a eukaryotic cell, and preferably a cell or cell line capable of being grown in vitro. Suitable cell lines can include, for example, CV-1 cells, COS cells, yeast or spodoptera cells. The invention also contemplates cells that can be prokaryotic in origin.
Suitable methods for isolating particular cells or tissues are 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 Ca2+, K+, and H+ ions, cell uptake of compounds such as stains, markers expressed on the cell surface, protein fluorescence, and membrane potential. Suitable methods that can be used in this regard include surgical removal of tissue, flow cytometry techniques such as fluorescence-activated cell sorting (FACS), immunoaffinity separation (e.g., magnetic bead separation such as Dynabead™ separation), density separation (e.g., metrizamide, Percoll™, or Ficoll™ gradient centrifugation), and cell-type specific density separation.
In an alternate embodiment, progenitor cells or tissues can be used for initially introducing the synthetic construct. Any suitable progenitor cell or tissue can be used which gives rise to a particular cell or tissue of interest for which codon preference is to be ascertained. For example, a suitable progenitor cell can comprise an undifferentiated cell. In the case of a plant, a suitable progenitor cell and tissue can include a meristematic cell and a callus tissue, respectively.
In another embodiment, the synthetic construct can be introduced first into an organism or part thereof before subsequent expression of the construct in a particular cell or tissue type. Any suitable organism is contemplated by the invention including unicellular and as multi-cellular organisms. Exemplary multi-cellular organisms include mammals (e.g. humans) and plants.
The invention further provides a synthetic construct comprising a reporter polynucleotide fused in frame with a tandem repeat of (e.g., 2, 3, 4, 5, 6, or 7 or more) identical codons, wherein said reporter polynucleotide encodes a reporter protein, and wherein said synthetic construct is operably linked to one or more regulatory polynucleotides.
The construction of the synthetic construct can be effected by any suitable technique. For example, in vitro mutagenesis methods can be employed, which are known to those of skill in the art. Suitable mutagenesis methods are described for example in the relevant sections of Ausubel, et al. (supra) and of Sambrook, et al., (supra) which are incorporated herein by reference. Alternatively, suitable methods for altering DNA are set forth, for example, in U.S. Pat. Nos. 4,184,917, 4,321,365 and 4,351,901, which are incorporated herein by reference. Instead of in vitro mutagenesis, the synthetic polynucleotide can be synthesized de novo using readily available machinery. Sequential synthesis of DNA is described, for example, in U.S. Pat. No. 4,293,652, which is incorporated herein by reference. However, it should be noted that the present invention is not dependent on, and not directed to, any one particular technique for constructing the synthetic construct.
Regulatory polynucleotides which can be utilized to regulate expression of the synthetic polynucleotide include, but are not limited to, a promoter, an enhancer, and a transcription terminator. Such regulatory polynucleotides are known to those of skill in the art. The construct preferably comprises at least one promoter.
Regulatory polynucleotides which can be utilized to regulate expression of the synthetic construct include, but are not limited to, a promoter, an enhancer, and a transcriptional terminator. Such regulatory polynucleotides are known to those of skill in the art. Suitable promoters that can be utilized to induce expression of the polynucleotides of the invention include constitutive promoters and inducible promoters.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
3.2. Determination of Abundance of Different tRNA Species in and/or Between Different Cells
The present invention contemplates any suitable method for determining the abundance of different iso-tRNA species in and/or between different cell or tissue types. For example, such method can include isolating a particular cell or tissue from a mammal, preparing an RNA extract from the cell or tissue which extract includes tRNA, and probing the extract with polynucleotides having different nucleic acid sequences, each being specific for a particular iso-tRNA to thereby determine the relative abundance of different iso-tRNAs in said cell or tissue. Preferably, this method is applied to two or more different cell or tissue types to determine the relative abundance of different iso-tRNAs between those cell or tissue types.
Suitable methods for isolating particular cells or tissues are known to those of skill in the art and are described, for example, in Section 3.1 above.
Any suitable method for isolating total RNA from a cell or tissue can 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 within the realm of one of ordinary skill in the art based on any known iso-tRNA sequence. Reference can be made in this regard to a compilation of tRNA sequences and sequences of tRNA genes described in Sprinzl et al (1996, Nucleic Acids Res. 24(1): 68-72; 1998, 26(1): 148-53; 20 Dec., 1999 http://www.uni-bayreuth.de/departments/biochemie/trna/), the entire disclosures of which are incorporated herein by reference.
In the case of a mouse, for example, exemplary oligonucleotide sequences which can be used include those described by Gauss and Sprinzl (1983, Nucleic Acids Res. 11: 1 incorporated herein by reference). In this respect, the oligonucleotide sequences can, for example, be any of:
Typically, the abundance of iso-tRNA species can be determined by blotting techniques that include a step whereby a 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 can be used to identify an RNA sequence that is complementary to a polynucleotide probe. Alternatively, dot blotting and slot blotting can be used to identify complementary DNA/RNA or RNA/RNA nucleic acid sequences. Such techniques are known by those skilled in the art, and have been described, for example, 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 polynucleotide (such as one having a sequence mentioned above) which is labeled, for example, radioactively, enzymatically or fluorochromatically.
While stringent washes are typically carried out at temperatures from about 42° C. to 68° C., one skilled in the art will appreciate that other temperatures can be suitable for stringent conditions. Maximum hybridization typically occurs at about 20° to 25° below the Tm for formation of a DNA-DNA hybrid. It is known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotides dissociate. Methods for estimating Tm are known in the art (see, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY supra at page 2.10.8). Maximum hybridization typically occurs at about 10° to 15° below the Tm for a DNA-RNA hybrid.
Other stringent conditions are known in the art. A skilled artisan 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 polynucleotides hybridized to an immobilized polynucleotide are known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection.
Advantageously, the relative abundance of an iso-tRNA between two or more cells or tissues can be determined by comparing the respective levels of binding of a labeled polynucleotide 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 between 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 can be selected such that the or each synonymous codon corresponds to an iso-tRNA which is in higher abundance in the target cell or tissue relative to other cells or tissues of the mammal.
In the present embodiment, a synonymous codon is preferably selected such that its corresponding iso-tRNA is present in the 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 present in one or more other cells or tissues of the mammal.
3.3. Analysis of Codon Usage
Alternatively, synonymous codons can be selected by analyzing the frequency at which codons are used by genes expressed in (i) particular cells or tissues, (ii) substantially all cells or tissues of the mammal, or (iii) an organism which can 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 Sharp et al (1988, Nucleic Acids Res. 16 8207-8211), which is incorporated herein by reference. Reference also can be made to an article by Nakamura et al (2000, Nucleic Acids Res 28(1): 292, incorporated herein by reference), which presents the frequency of each of the 257 468 complete protein-coding sequences (CDSs) compiled from the taxonomic divisions of the GenBank DNA sequence database. The sum of the codons used by 8792 organisms is also calculated. The data files relating to this article can be obtained from the anonymous ftp sites of DDBJ, Kazusa and EBI. A list of the codon usage of genes and the sum of the codons used by each organism can, for example, be obtained through the web site http://www.kazusa.or.jp/codon/.
The relative level of gene expression (e.g., detectable protein expression vs. no substantial or detectable protein expression) can provide an indirect measure of the relative translational efficiencies of codons, the relative abundance of specific iso-tRNAs expressed, or both, in different cells or tissues. For example, a virus can be capable of propagating within a first cell or tissue (which can include a cell or tissue at a specific stage of differentiation) but can be substantially incapable of propagating in a second cell or tissue (which can 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 can thus provide indirectly a set of codons that have high translational efficiencies and a set of codons that have low translational efficiencies in the first cell or tissue relative to the second cell or tissue.
Simultaneously, the above comparison can also provide indirectly a set of codons that that have higher translational efficiencies and a set of codons that have low translational efficiencies in the second cell or tissue relative to the first cell or tissue.
From the foregoing, a synonymous codon according to the invention can correspond to a codon selected from the group consisting of (1) a codon used at relatively high frequency by genes, preferably highly expressed genes, of a target cell or tissue, (2) a codon used at relatively high frequency by genes, preferably highly expressed genes, of the mammal, (3) a codon used at relatively low frequency by genes of one or more other cells or tissues, and (4) a codon used at relatively low frequency by genes of another organism.
By contrast, an existing codon according to the invention can correspond to a codon selected from the group consisting of (a) a codon used at relatively high frequency by genes, preferably highly expressed genes, of one or more other cells or tissues, (b) a codon used at relatively low frequency by genes of a target cell or tissue, (c) a codon used at relatively low frequency by genes of the mammal, and (d) a codon used at relatively high frequency by genes of another organism.
Preferably, the genes from which codon frequency data are obtained do not relate to mitochondrial genes.
Suitably, a highly expressed gene according to the invention encodes a protein that is expressed at high levels, and preferably specifically (i.e., substantially only, e.g. at a level at least about 100-fold greater than in other cells or tissues), in the target cell/tissue. Examples of such genes include the lactalbumen gene expressed in breast epithelium, oncogenes expressed in cancer cells (e.g., Bcl-2, p53, erbB, C-myb, C-mos, C-rel etc), insulin expressed in beta cells, transglutaminase and loricrin expressed in differentiated epithelium, and E2F1 expressed in cycling undifferentiated cells.
Codons used at a relatively high frequency by genes, preferably highly expressed genes, of the mammal can 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), agc (Ser), ucu (Ser), ucc (Ser), and uca (Ser). Alternatively, such codons can 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 mammals are described, for example, in Sharp et al (1988, supra). Such codons include, but are not restricted to, cua (Leu); cga (Arg), cgu (Arg); ucg (Ser). Alternatively, such codons can include aua (Ile); gua (Val); acg (Thr); gcg (Ala); caa (Glu); ggu (Gly).
4. Construction of Synthetic Polynucleotides
The step of replacing synonymous codons for existing codons can be effected by any suitable technique. For example, in vitro mutagenesis methods can be employed which are known to those of skill in the art and include those described in Section 3.1 above.
The parent polynucleotide is preferably a natural gene. However, it is possible that the parent polynucleotide encodes a protein that is not naturally-occurring but has been engineered using recombinant techniques.
The parent polynucleotide need not be obtained from the mammal but can be obtained from any suitable source, such as from a eukaryotic or prokaryotic organism. For example, the parent polynucleotide can be obtained from another mammal or other animal. Alternatively, the parent polynucleotide can 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 can be a yeast, bacterium or virus.
For example, suitable proteins which can 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 polynucleotide encoding the CFTR protein which can be utilized to produce the synthetic polynucleotide is described, for example, in Riordan et al (1989, Science 245 1066-1073), and in the GenBank database under locus designation HUMCFTRM (Accession number M28668), which are incorporated herein by reference.
Regulatory polynucleotides which can be utilized to regulate expression from the synthetic polynucleotide include, but are not limited to, a promoter, an enhancer, and a transcriptional terminator. Such regulatory polynucleotides are known to those of skill in the art. Synthetic polynucleotides according to the invention can be operably linked to one or more regulatory polynucleotides in the form of an expression vector.
The invention also contemplates synthetic polynucleotides encoding one or more desired portions of the protein to be expressed. A polynucleotide encodes a domain of the protein having a function associated therewith, or which is otherwise detectable, and preferably encodes at least 10, 20, 50, 100, 150, or 500 contiguous amino acid residues of the protein.
The step of introducing the synthetic polynucleotide into a target cell differs depending on the intended use and species, and can involve one or more of non-viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R. C., (1993 Science 260 926-932) which is hereby incorporated by reference. Such methods can include, for example:
A. Local application of the synthetic polynucleotide 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 can 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 polynucleotide so as to increase the effectiveness of that treatment. This method can 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.
B. General systemic delivery by injection of DNA, (Calabretta et al., 1993, Cancer Treat. Rev. 19 169-179, which is incorporated herein by reference), or RNA, alone or in combination with liposomes (Zhu et al., 1993, Science 261 209-212, which is incorporated herein by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is incorporated herein by reference) or any other mediator of delivery. Improved targeting might be achieved by linking the synthetic polynucleotide 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 encoding said synthetic polynucleotide, or of cells responsive to said protein.
C. 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 incorporated herein by reference), infection, injection, electroporation (Shigekawa et al., 1988, BioTech. 6 742-751, which is incorporated herein by reference) or any other way so as to increase the expression of said synthetic polynucleotide in those cells. The modification can 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 incorporated herein by reference) or other vectors, or other agents of modification such as liposomes (Zhu et al., 1993, Science 261 209-212, which is incorporated herein by reference), viral capsids or nanoparticles (Bertling et al., 1991, Biotech. Appl. Biochem. 13 390-405, which is incorporated herein 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 incorporated herein by reference. Treated cells can be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.
5. Pharmaceutical Compositions
In yet another aspect, the invention provides a pharmaceutical composition comprising the synthetic nucleic sequences of the invention and a pharmaceutically acceptable carrier.
Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, known in the art can be used. These carriers can 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 can be employed for determining expression of the protein from said synthetic polynucleotide 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 known to those skilled in the art.
6. Applications
6.1. Targeting Expression of a Protein to a Differentiated Cell
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 polynucleotide at a level 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, when compared to the at least one existing codon, the synonymous codon has a higher translational efficiency in the differentiated cell compared to the precursor cell. Accordingly, a synthetic polynucleotide is produced having altered translational kinetics compared to the parent polynucleotide 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.
The above embodiment can 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 can include cystic fibrosis transmembrane conductance regulator (CFTR) protein, and adenosine deaminase (ADA).
The differentiated cell can comprise a cell of any lineage including a cell of epithelial, hemopoetic or neural origin. For example, the differentiated cell can be a mature differentiated keratinocyte.
6.2. Targeting Expression of a Protein to Progeny of a Stem Cell But not to the Stem Cell Itself
The synthetic polynucleotide produced above can 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 can result in loss of stem phenotype that is undesirable. However, an advantageous therapy can reside in transducing autologous marrow stem cells with a synthetic polynucleotide operably linked to one or more regulatory polynucleotides, wherein existing codons of the wild type ADA gene have been replaced with synonymous codons which have higher translational efficiencies in differentiated lymphocytes compared to the marrow stem cells. The transduced stem cells can then be re-infused 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 can comprise stem cells isolated as CD34 positive cells from a patient's peripheral blood or marrow. For gene delivery, a suitable vector can include a retrovirus or an adeno-associated virus.
Alternatively, in the case of inducing cell mediated immunity, dendritic cells are important 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 can not be optimal. However, in accordance with the present invention, a long-term immune stimulation can be provided by transducing autologous bone marrow-derived CD34 positive dendritic cell precursors with a synthetic polynucleotide encoding an antigen, such as the melanoma antigen MART-1. In this example, the synthetic sequence is constructed such that existing codons of a wild-type polynucleotide encoding MART-1 are replaced with synonymous codons which have higher translational efficiencies in the dendritic cells compared to the dendritic cell precursors. The transduced dendritic cell precursors can then be re-infused 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 re-stimulation of a cytotoxic T lymphocyte (CTL) response to the MART-1 antigen.
6.3. Targeting Expression of a Protein to a Stem Cell But not to Progeny of the Stem Cell
In an alternate embodiment, the target cell can be an undifferentiated cell wherein the protein is not expressible in said undifferentiated cell from a parent polynucleotide 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 polynucleotide is replaced with a synonymous codon which has a higher translational efficiency in the undifferentiated cell compared to a differentiated cell. This results in a synthetic polynucleotide having altered translational kinetics compared to said parent polynucleotide 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 existing codons have low translational efficiencies in the stem cell and that therefore the protein is not normally capable of being expressed at levels sufficient to commit the stem cell to differentiate along a particular cell lineage. However, upon delivery of a parent polynucleotide encoding the protein and having altered codons (i.e. one or more first codons replaced with more highly efficiently translated synonymous codons), the protein can be expressed in the undifferentiated stem cell, thereby inducing differentiation of the stem cell into a desired cell lineage. It will also be apparent that such commitment to differentiate along a particular cell lineage can be utilized to prevent production of a particular lineage of cells such as cancer cells.
Alternatively, the method according to this embodiment can be used to express a transcriptional regulatory protein that is involved in the production of a therapeutic agent or agents. Such a protein can include, for example, NF-kappa-B transcription factor p65 subunit (NF-kappa-B p65) which is involved in the production of interleukin-2 (IL-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 having low translational efficiencies in stem cells. Accordingly, such a sequence can be used as the sequence of a parent polynucleotide 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 GenBank database as locus HUMNFKB65A, having Accession Number L19067, which are incorporated herein by reference.
A suitable undifferentiated cell which can 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 can also be used advantageously for gene therapy where ongoing regulated expression of a transgene is desirable. For example, secure but reversible induction of fertility is desirable in veterinary practice and in humans. Such induction can be effected by transducing autologous breast ductal epithelial cells with a synthetic polynucleotide encoding a leutinising hormone (LH) antagonist or a leutinising hormone releasing hormone (LHRH) antagonist under the control of one or more regulatory polynucleotides. The synthetic polynucleotide can be produced by replacing existing codons of a parent polynucleotide with synonymous codons having high translational efficiencies in resting breast ductal epithelial cells compared to differentiated cells arising therefrom. Once the transduced cells are implanted back into the patient, expression can 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. In one example of determining codon translational efficiencies according to this embodiment, the iso-tRNA composition of resting and estrogen-deprived breast epithelial cells can be established by first obtaining resting cells from reduction mammoplasty, and determining the cellular tRNA composition in the presence and absence of estrogen. The synthetic polynucleotide can be introduced into autologous resting epithelial cells by cell electroporation ex vivo, and the transduced cells can be subsequently transplanted subcutaneously into the patient. Progestagen can be administered as required to reverse induction of fertility.
6.4. 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 are generally toxic for all dividing cells. This problem can be nevertheless ameliorated by establishing the iso-tRNA composition in a tumor clone, and constructing a synthetic toxin gene (e.g., ricin gene) or a synthetic anti-proliferation gene (e.g., the tumor suppressor 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. Alternatively, synonymous codons can be deduced from an analysis of the translational efficiencies of different codons in tumor cells compared to normal dividing cells to thereby construct the synthetic gene. 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 (i.e., a drug resistance gene e.g., the multi-drug resistance gene) having a codon pattern unlikely to be expressed efficiently in the tumor can be employed.
6.5 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, leptin-induced starvation can occur. Advantageously, a synthetic gene encoding leptin can be constructed including synonymous codons having high translational efficiencies in activated adipocytes compared to non-activated adipocytes. The synthetic gene can then be introduced into the patient by suitable means such that leptin is expressed in activated adipocytes and not expressed (or not substantially expressed) in non-activated adipocytes. As body fat turnover diminishes under the influence of leptin-reduced appetite, the metabolic activity of the adipocytes falls and the leptin production decreases correspondingly.
6.6. Targeting Expression of a Protein to a Stage of the Cell Cycle
In another embodiment of the invention, the target cell can 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 polynucleotide at a level sufficient to effect a particular function associated with the protein. The synonymous codons are selected such that each has a higher translational efficiency in the non-cycling cell compared to the cycling cell. Accordingly, a synthetic polynucleotide is produced having altered translational kinetics compared to the parent polynucleotide 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.
6.7. Expressing a Protein in a Target Cell or Tissue by In Vivo Expression of Iso-tRNAs in the Target Cell or Tissue
The invention also extends to a method wherein a protein can be selectively expressed in a target cell by introducing into the cell an auxiliary polynucleotide capable of expressing in the target cell one or more isoaccepting transfer RNAs which are not normally expressed in relatively high abundance in the target cell but which are rate-limiting for expression of the protein from a parent polynucleotide to a level sufficient for effecting a function associated with the protein. In this embodiment, introduction of the auxiliary polynucleotide sequence in the target cell changes the translational kinetics of the parent polynucleotide such that said protein is expressed at a level sufficient to effect a function associated with the protein.
The step of introducing the auxiliary polynucleotide sequence into the target cell or a tissue comprising a plurality of these cells can be effected by any suitable means. For example, analogous methodologies for introduction of the synthetic polynucleotide referred to above can be employed for delivery of the auxiliary polynucleotide into said target cell.
In practice, the choice of iso-tRNA supplied to a target cell using this method depends on whether the target cell in which protein expression is desired has a low or high abundance of that iso-tRNA and on whether the parent polynucleotide comprises codons corresponding to that iso-tRNA species. Thus, an iso-tRNA is supplied to the target cell by the auxiliary polynucleotide when that iso-tRNA is in relatively low abundance in the target cell and when parent polynucleotide comprises codons corresponding to that iso-tRNA species.
8. Assembly of Virus Particles in Cells which do not Normally Permit Assembly of Virus Particles
The invention also provides a method for producing a virus particle in a cycling eukaryotic cell. The virus particle comprises at least one protein necessary for virus assembly, wherein the at least one protein is not expressed in the cell from a parent polynucleotide at a level sufficient to permit virus assembly therein. This method is characterized by replacing at least one existing codon of the parent polynucleotide with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to the parent polynucleotide such that the at least one protein is expressible from the synthetic polynucleotide in the cell at a level sufficient to permit virus assembly therein. The synthetic polynucleotide so produced is operably linked to one or more regulatory polynucleotides 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 codons, when compared to their corresponding existing codons, have higher translational efficiencies in the cycling cell compared to a non-cycling cell.
The cycling cell can be any cell in which the virus is capable of replication. Suitably, the cycling cell is a eukaryotic cell. Preferably, the cycling cell used 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 is a viral capsid protein or capsomer. Suitable viral capsid proteins include, but are not restricted to, the L1 and/or L2 proteins of papillomavirus, VP1-3 of polyomavirus, VP1-6 of blue tongue virus, and the capsid proteins of adenovirus.
The other viral proteins required for assembly of the virus particle in the cell can be expressed from one or more other polynucleotides 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 polynucleotide(s) preferably comprises the papillomavirus genome without the polynucleotides encoding L1 and/or L2.
In another embodiment, there is provided a method for producing a virus particle in a cycling cell wherein the virus particle comprises at least one protein that is necessary for assembly of the virus particle. In this embodiment, the protein is not expressed in the cycling cell from a parent polynucleotide at a level sufficient to permit virus assembly therein, and at least one existing codon of the parent polynucleotide is rate-limiting for the production of the at least one protein. The method includes introducing into the cell a polynucleotide capable of expressing therein an isoaccepting transfer RNA specific for said at least one codon.
The invention also provides virus particles made by any of the above methods, as well as cells or tissues containing therein the synthetic polynucleotides 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.
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 made when genes from prokaryotic organisms have been expressed in eukaryotic cells (Smith, D. W., 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 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 1). An apparent exception is that PV L1 genes can be efficiently expressed in insect cells (Kirnbauer, et al., 1992, 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, 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 exhibit 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 iso-tRNAs for that organism (Ikemura, T., 1981, J. Mol. Biol. 146:1-21). However, populations of tRNAs in differentiating and neoplastic cells are different (Kanduc, D., 1997, Arch. Biochem. Biophys. 342:1-6; 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 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. N., et al.), Lippincott-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 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 (
Because inhibitory RNA sequences within the L1 coding sequence could have been rendered non-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 polynucleotides 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 GenBank. 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 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 60-80% 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, 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 requires comparisons of transcriptional activity and translation of the L1 or L2 genes driven from strong constitutive promoters in differentiated and undifferentiated epithelium. Such work can be performed 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. 71:5161), and the data presented here indicate that translation regulation can 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 specialized 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.
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:1,2) and L2 genes (SEQ ID NOS:5,6) are shown respectively in
For immunoblotting assays, COS-1 cells in 6-well plates were transfected with 2 μg L1 or L2 expression plasmids using lipofectamine (Gibco). 36 hrs after transfection, cells were washed with 0.15M phosphate buffered 0.9% NaCl (PBS) and lysed in SDS loading buffer. The cellular proteins were separated by 10% SDS PAGE and blotted onto a 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 36 hr 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 PGFP plasmid transfected cells, the cell were fixed with 4% buffered formaldehyde and viewed by epi-fluorescence microscopy.
Northern Blotting
COS-1 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 MgCl2 and 0.5% NP40) was directly added to monolayer cells and cells were lysed in 4° C. for 5 min. After the nuclei were removed by centrifugation, cytoplasmic RNA was purified by column chromatography. 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 1.5% agarose gel in the presence of formaldehyde. The RNAs were then blotted onto nylon membrane and probed with (a) 1:1 mixed 5′-end labeled L1 wt and HBL1 fragments; (b) 1:1 mixed 5′-end labeled L2 wt and HBL2 fragments; (c) 1:1 mixed 5′-end labeled GFP and PGFP fragments or (d) randomly labeled PAGDH fragment. The blots were washed extensively at 65° C. 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 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 (
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 (
To establish whether the alterations to the primary sequence of the L1 and L2 mRNA that 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 (
In a comparative study, the wild-type and humanized L1 gene were also transfected into five laboratory cell lines representative of different tissue types (CV-1, COS 1, 3T3, HL-60 and pTET). As expected from the above results, the humanized L1 gene is well expressed in CV-1 cells (of epithelial origin), and in COS-1 cells, which are similar in origin, whereas the wild-type gene is not detectably expressed in these cells. Both constructs were also found to be poorly expressed in undifferentiated HL-60 cells (of hemopoetic origin), and in 3T3 cells (of mesenchymal origin), whereas the wild-type gene but not the humanized gene was expressed in pTET.
Papillomavirus Late Protein Translation In Vitro
Materials and Methods
In Vitro Translation Assay
One microgram of each plasmid was incubated with 20 μCi 35S-methionine (Amersham) and 40 μL 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
2.5×10−4 M tRNA (Boehringer) was added to a 20 μL reaction containing 10 mM Tris-acetate, pH.7.8, 44 mM KCl, 12 mM MgCl2, 9 mM-mercaptoethanol, 38 mM ATP, 0.25 mM GTP and 7 μL rabbit reticulocyte extract. The reaction was carried out at 25° C. for 20 min, and 30 μL H2O was added to the reaction to dilute the tRNAs to 1×10−4 M. The aminoacyl-tRNAs were then aliquoted and stored at −70° C.
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 intact cells can 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 35S-methionine. After 20 minutes, translated proteins were separated by SDS PAGE and visualized by autoradiography. Efficient translation of the modified L1 gene was observed (
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 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 (
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 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 (
Substitution of papillomavirus-preferred codons prevents translation but not transcription of a non-papillomavirus 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
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 (
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 25 μg 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 (
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 μg/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° C. and examined for expression of L1 gene by standard immunofluorescence methods using BPV L1-specific antibody and quantified by FACS analysis (Qi et al 1996, Virology 216 35-45).
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 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 can 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 can be tested in focus formation assays using C127 fibroblasts.
Analysis of tRNA Composition
Method for Extracting and Measuring tRNA from Tissues
Tissue (100 g) 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 NaCl, 0.005 M EDTA in 0.1 M Tris-chloride 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 95% ethanol were added. The resultant precipitate was spun down at top speed in the International™ clinical centrifuge and re-suspended 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 (2×10 cm) of 2 g of DEAE-cellulose previously equilibrated with cold 0.1 M Tris-chloride buffer pH 7.5. The column was then washed with 1 L of Tris-chloride 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 10 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 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° C. for 15 min in 1-5 μL of 15% formaldehyde. 10×SSC (SSC is NaCl 0.3 M, tri-sodium citrate 0.03 M). The samples are spotted in 1 μL aliquots onto the membranes that have been soaked for 15 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 ultraviolet-irradiation (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° C.
A 5′ end labeled synthetic deoxyribo-oligonucleotide complementary to the A54-A73 sequence of the tRNA is used as a probe for the hybridization experiments. Labeling of the oligonucleotide is performed by direct phosphorylation of the 5′-OH ended probe.
For hybridization experiments, the UV-irradiated membranes are first pre-incubated for 5 h at 50° C. in 50% deionized formamide, 5×SSC, 1% SDS, 0.04% Ficoll™ 0.04% polyvinylpyrrolidone and 250 μL/mL of sonicated salmon sperm DNA using 5 mL of buffer for 100 cm2 of membrane. Hybridization is finally performed overnight at 50° C. in the above solution (2.5 mL/100 cm2) where the labeled probe has been added. After hybridization, the membranes are washed twice in 2×SSC, 0.1% SDS for 5 min at room temperature, twice in 2×SSC, 1% SDS for 30 mm at 60° C. and finally in 0.1×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
The sequences of the tRNA probes are as follows:
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° C. 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 re-suspended in 30% Percoll and separated by centrifugation through a discontinuous Percoll gradient (1.085, 1.075 and 1.050 g/mL) at 1200×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 5.5). After centrifugation, the supernatant was diluted with 3 volumes of 100 mM Tris (pH 7.5) and added to a DEAE column equilibrated with 100 mM Tris (pH 7.5). An equal volume of isopropanol was added to the aqueous solution containing tRNA, and the solution was allowed to stand overnight at 4° C. The tRNA was spun down and washed with 75% ethanol, then dissolved in RNase-free water.
tRNA Blotting
Ten nanograms of each tRNA sample in 1 μL was denatured in 60° C. for 15 min in 4 μL formaldehyde and 5 μL 20×SSC. The samples were spotted in 1 μL aliquots onto charged nylon membrane (Amersham), and the tRNAs were fixed with UV and probed with 32P-oligonucleotides.
Results
Comparison of the abundance of the tRNA species in undifferentiated and differentiated keratinocytes showed that the levels of some tRNA populations changed dramatically. For example, the levels of tRNAs specific for AlaGCA, LeuCTT, LeuCTA were increased in differentiated cells while tRNAs for ArgCGA, ProCCI, AsnAAC were more abundant in undifferentiated keratinocytes (see Table 2).
Construction of expression vectors for determining relative codon preferences in mammalian cells.
Synthetic gfp genes were constructed in which a single artificial start codon (ATG) followed by a stretch of five identical codons is fused in frame immediately upstream of a gfp coding sequence. A reverse oligonucleotide primer (SEQ ID NO:219; sequence complementary to the termination codon for GFP, is underlined), and a suite of forward oligonucleotide primers (SEQ ID NO: 160 through 218; the first codon of GFP, is underlined) were synthesized and used for PCR amplification of a humanized gfp gene (SEQ ID NO:158) (GIBCO) as template with Taq DNA polymerase (Amplification parameters: 95° C./30 sec; 52° C./30 sec; 72° C./1 min; 30 cycles). The amplified fragments have nucleic acid sequences and deduced amino acid sequences as shown in SEQ ID NO:35 through 157.
In summary, the synthetic fragments contain an artificial start codon followed by a tandem repeat of five identical codons specific for a given iso-tRNA species. The tandem repeat immediately precedes the second codon of the gfp gene. The synthetic fragments by SEQ ID NO, and encoded tandem repeat, are presented in the TABLE 3.
The amplified fragments were cloned between the EcoRI and KpnI sites of the mammalian expression vector pCDNA3 containing SV40 ori (Invitrogen) and the CMV promoter.
Transfection of COS-1 Cells
COS-1 cells were grown continuously in DMEM media supplemented with 10% fetal calf serum (FCS), glutamine, penicillin and streptomycin. Cells were passaged from a 150 cm2 flask into multiple 25 cm2 flasks. Cells were transfected using a QIAGEN Effectene™ transfection kit (and the manufacturer's instructions, incorporated herein by reference) when confluency of the cells was between 60-80%. Briefly, 1 μg of plasmid DNA was diluted into 10 μL of filtered TE buffer and 140 μL of QIAGEN™ Buffer EC. Eight microliters of QIAGEN™ Enhancer was added followed by vortexing and incubation at room temperature for 2-5 min. QIAGEN™ Effectene (10 μL) was added followed by vortexing for 10 seconds and a further incubation at room temperature for 10 min. The cells were washed once in 1×PBS followed by re-suspension in fresh media (1 mL). After 48 hrs, cells were harvested and washed in 1×PBA (phosphate buffered saline plus azide). Cells adhering to the flask were removed by scraping with a cell scraper. Cells were then filtered through a 70 μm filter before addition of 300 μL of 2% paraformaldehyde and 300 μL of 10×FCS. Cells were kept on ice in the dark until FACS analysis.
Synthetic gfp mRNA expression of transfected cells was tested by reverse transcriptase PCR. GFP protein expression was analyzed by confocal microscopy and flow cytometry.
Confocal Microscopy
Transfected COS-1 cells were examined using a Bio-Rad MRC-600 laser-scanning confocal microscope equipped with a krypton-argon laser and filter sets suitable for the detection of fluorescein and Texas red dyes (Bio-Rad K1yK2), and a Nikon 603 PlanApo™ numerical aperture 1.2 water-immersion objective. Dual-channel confocal images and video montages of the transfected cells can be suitably composed using ADOBE PhotoShop™.
Flow Cytometry
Transfected COS-1 cells were analyzed with a Becton Dickinson™ Flow cytometer Elite II. Omega Filters™ allowed detection of green fluorescence emission (EMI510/20—collects light from 490-530 nm) and yellow fluorescence emission (EM2 550/30—collects light form 525-580 nm) from the transfected cells.
Results
A series of 64 reporter constructs (see TABLE 3) was made and validated, in which the gfp gene is preceded in frame by a tandem repeat of 5 identical codons. Together, the series covers the entire set of isoaccepting codon triplets.
The series was transfected into a single cell line, and expression levels measured by flow cytometry (see TABLE 4). Overall, the expression level of the reporter gene constructs in the cell line varied over a range of 20-fold, according to the triplet used in the reporter construct. Repeated determinations on the same construct showed excellent inter-assay reproducibility (r2=0.9).
Variation in expression levels across the isoaccepting codons for a single amino acid ranged from 1.4-fold for valine to 13-fold for threonine, with a median of about 4-fold. Variation in expression between amino acids was of the same order of magnitude. The order of magnitude of the effect is defined as an average of 4 fold per amino acid if 5 copies are incorporated, compatible with an extreme in range of expression levels of up to (1.6)200=1086 over an average 200-amino acid residues protein. This figure is derived as: [1+((4−1)(range of reporter construct expression)/5 (no of triplets in the reporter construct))]200
The results presented in TABLE 4 also show that various codons in the undifferentiated epithelial cells (COS-1) have translational efficiencies at least two-fold higher or two-fold lower relative to those of their corresponding synonymous codons. Representative codons having at least a two-fold higher translational efficiency relative to at least one of their corresponding synonymous codons include aga (Arg), cgg (Arg), tgc (Cys), gga (Gly), ggc (Gly), ccg (Pro), cga (Pro), aca (Thr), acg (Thr), and act (Thr). Thus, these codons appear to be preferred for translation in the undifferentiated epithelial cells. By contrast, representative codons having at least a two-fold lower translational efficiency relative to at least one of their corresponding synonymous codons include agg (Arg), tgt (Cys), ggg (Gly), ggt (Gly), ccc (Pro), cct (Pro), and acc (Thr). These latter codons would therefore appear to be less preferred for translation in the undifferentiated epithelial cells. Accordingly, if higher protein expression is required within undifferentiated epithelial cells such as COS-1 cells, the preferred codons should be used to replace any existing codons of a parent polynucleotide encoding the protein that correspond to the less preferred codons. In this respect, a codon substitution algorithm for increasing protein expression in non-differentiated epithelial cells is presented in TABLE 5. However, if lower protein expression is required in non-differentiated epithelial cells, the less preferred codons should be used to replace any existing codons of the parent polynucleotide that correspond to the preferred codons.
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety.
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 can 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.
Table Legends
Table 1
The codon usage data for human, cow, yeast and wheat proteins are derived from published results (Wada, K. et al, 1992, Nucleic Acids Res 20 Suppl: 2111-2118; 1991, Nucleic Acids Res 19 Suppl: 1981-1986; 1990, Nucleic Acids Res 18 Suppl: 2367-411), which are incorporated herein by reference. The BPV1 data are from sequences in the GenBank database.
Table 2
Each iso-acceptor tRNA with anticodon shown as superscript is shown on top row. The letter “x” indicates an arbitrary value for the relative abundance of tRNA between differentiated (“superficial” layer of the epidermis) and undifferentiated (“basal” layer of the epidermis) cell layers, wherein “10×” and “100×” indicates about a ten-fold and about a one hundred-fold difference, respectively, in abundance of iso-tRNA between these layers.
Table 3
Synthetic gfp constructs are tabulated by SEQ ID NO and by the codon corresponding to the tandem repeat of five identical codons immediately upstream of the gfp gene.
Table 4
Mean fluorescence intensities of up to four different samples of transiently transfected COS-1 cells are shown (Green mean 1-4). Synthetic gfp constructs are tabulated by SEQ ID NO and by the codon corresponding to the tandem repeat immediately upstream of the gfp gene.
Table 5
Input codons and output codons represent, respectively, synonymous codons and existing (i.e. “first”) codons according to the invention. Change means an actual change of a codon.
Tables
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PO7765 | Jul 1997 | AU | national |
| PO9467 | Sep 1997 | AU | national |
| PP8078 | Jan 1999 | AU | national |
This application is a divisional of U.S. patent application Ser. No. 10/305,633, filed Nov. 27, 2002, now abandoned which is a divisional of U.S. patent application Ser. No. 09/479,645, filed Jan. 7, 2000, now U.S. Pat. No. 6,489,141 B1, issued Dec. 3, 2002, which claims priority of Australian Patent Application No. PP 8078, filed Jan. 8, 1999, and which is a continuation-in-part of co-pending International Patent Application No. PCT/AU98/00530, filed Jul. 9, 1998, which was published in English and designates the United States, and which claims priority of Australian Patent Application Nos. PO7765, filed Jul. 9, 1997, and PO9467, filed Sep. 11, 1997. The subject matter of related applications PCT/AU98/00530, U.S. Ser. No. 09/479,645 and U.S. Ser. No. 10/305,633 are hereby incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6489141 | Frazer et al. | Dec 2002 | B1 |
| 7229761 | Frazer et al. | Jun 2007 | B2 |
| Number | Date | Country |
|---|---|---|
| 9609378 | Mar 1996 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20070050859 A1 | Mar 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10305633 | Nov 2002 | US |
| Child | 11591969 | US | |
| Parent | 09479645 | Jan 2000 | US |
| Child | 10305633 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/AU98/00530 | Jul 1998 | US |
| Child | 09479645 | US |
