Patent Application
20100040649
The invention relates to molecular biology, in particular to improved expression systems of nucleic acids.
Systems to modulate nucleic acid expression are important for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, vaccination, animal models for human diseases and biopharmaceutical protein production. In these applications expression of a nucleic acid(s) of interest is preferably controlled in a quantitative and temporal way. Several artificial gene expression systems that are regulated by non-toxic effector molecules in a dose-dependent and reversible manner are currently available. The Tet system, in which gene expression is stringently controlled by tetracycline (Tc) or its derivative doxycycline (dox), is the most widely used regulatory circuit (Baron et al. 2000; Gossen et al. 2001; Berens et al. 2003). This system is based on the sequence-specific, high-affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Tc or dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO. Fusion of the VP16 activation domain of herpes simplex virus to TetR resulted in the transcriptional activator tTA, which induces nucleic acid expression from tetO-containing promoters (Ptet) in eukaryotic cells (Gossen et al. 1992). The presence of Tc or dox abolishes tTA-tetO interaction and switches off gene expression (Tet-off system). A tTA variant with four amino acid substitutions in the TetR moiety was identified, which exhibits a reverse phenotype (Gossen et al. 1995). This reverse tTA (called rtTA) binds to Ptet exclusively in the presence of dox, but not in its absence (Tet-on system). Both Tet systems are now widely applied to control nucleic acid expression in eukaryotes, including mammals, plants and insects (reviewed in (Gossen et al. 2001)). Because long-term exposure to effectors is often undesirable, the Tet-on system is preferred in applications in which nucleic acid expression is to be sustained in a switched-off state for long periods, or when rapid induction of nucleic acid expression is required.
Unfortunately, the amino acid substitutions in rtTA that confer the reverse phenotype also affect its binding affinity for effectors. As a consequence, rtTA has lost the ability to be activated by Tc and other Tc-like compounds, and it requires 100-fold more dox for maximal induction than that is needed for tTA inhibition. These characteristics severely limit the in vivo use of the Tet-on system. For example, to activate Tet-on controlled transgene expression in the rat brain, the animals have to be fed with high doses of dox that are nearly toxic (Baron et al. 1997). Therefore, the Tet-on system, particularly its effector-sensitivity, has to be improved.
Previously, the Tet system has been optimized by introduction of rationally designed mutations (Baron et al. 1997; Baron et al. 1999), and by directed evolution in which random mutagenesis of the components of the Tet system was followed by functional screening of the mutants in bacterial or yeast assay systems (Gossen et al. 1995; Urlinger et al. 2000). However, these approaches are labor intensive, and mutations selected in bacterial or yeast assay systems are not necessarily improvements in higher eukaryotes.
Another disadvantage of current rtTA systems is the risk of reduced dox-dependence after multiple rounds of replication. This problem for instance arises during vaccination applications where replication of at least part of a pathogen is under control of an rtTA system. In such vaccination applications, protection against said pathogen is acquired by controlled, inducible replication of said at least part of a pathogen, preferably during a restrained time span. If however the rtTA system looses its dox-dependence, said at least part of a pathogen will constitutively replicate, resulting in too much pathogenic nucleic acid and/or proteins, involving a safety problem. The same kind of problem arises during other applications involving various rounds of amplification of rtTA. It is therefore desired to improve the genetic stability of current rtTA systems.
It is an object of the present invention to provide rtTA and single chain rtTA variants. Preferably rtTA and single chain rtTA variants are provided with at least one improved property.
The invention provides a method for inducibly expressing a nucleic acid sequence of interest, the method comprising:
providing a nucleic acid construct comprising said nucleic acid sequence of interest operably linked to an inducible gene expression system which comprises an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising a mutation in a codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195;
introducing said nucleic acid construct to a suitable expression system; and
allowing for inducible expression of said nucleic acid sequence of interest.
According to the present invention, a mutation in at least one of the above mentioned codons of an rtTA nucleic acid or a single chain rtTA (sc rtTA) nucleic acid results in an improved rtTA or sc rtTA activator as compared to currently used Tet-on systems, such as described in (Gossen et al. 1995), (Urlinger et al. 2000) and (Krueger et al. 2003). Using an rtTA or sc rtTA variant of the present invention, an improved rtTA or sc rtTA system is provided which has a higher transcriptional activity, a higher dox-sensitivity, a higher genetic stability and/or a lower level of transcription in the absence of an inducer, as compared to currently used rtTA or sc rtTA systems. The level of transcription in the absence of an inducer is called herein basal activity. Furthermore, rtTA and sc rtTA systems are provided which are inducible by antibiotics other than doxycycline. Hence, the use of an rtTA and/or sc rtTA of the present invention is preferred for inducibly expressing a nucleic acid sequence of interest.
A preferred embodiment provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence further comprises a mutation in a codon at rtTA amino acid position 12, and/or 86, and/or 209. It has been shown that such additional mutations result in improved characteristics of the resulting rtTA and sc rtTA systems.
A single chain rtTA (sc rtTA) is a monomer comprising the same transregulating properties as the rtTA dimer in kind, not necessarily in amount. Said sc rtTA preferably comprises two TetR moieties and one eukaryotic regulatory domain. Said two TetR moieties are preferably connected to each other by a linker, said linker preferably comprising a sequence encoding an (SG4)5 linker which is long and flexible enough to allow intramolecular assembly of the two TetR proteins. Non-limiting examples of single-chain Tet transregulators are described in (Krueger et al. 2003). The methods described therein on page 3050, last paragraph and page 3051 for generating a single chain tTA and/or a single chain rtTA are incorporated herein by reference. These methods are non-limiting examples of generating sc rtTA.
According to the present invention, a mutation in a sc rtTA which corresponds to a mutation according to the present invention in an rtTA dimer transregulator results in an improved sc rtTA. A mutation in a sc rtTA corresponds to a mutation according to the invention in an rtTA dimer when a mutation in a sc rtTA is present in a codon encoding an amino acid residue at a position within said sc rtTA which is comparable to rtTA amino acid position 9, 12, 19, 37, 56, 67, 68, 86, 138, 157, 171, 177, 195 and/or 209.
By inducibly expressing a nucleic acid sequence of interest is meant herein that expression of a nucleic acid of interest is at least in part influenced by at least one inducer. Hence, by regulating the amount of inducer that is administered to said expression system, one is capable of regulating the amount of expression of said nucleic acid sequence of interest. Said inducer preferably comprises an exogenous compound, meaning that said compound is not naturally present within said expression system. Preferably, expression of a nucleic acid sequence of interest is dependent on the presence of an inducer. This means that said nucleic acid is expressed in the presence of an inducer, while it is expressed to a significant lesser extent in the absence of said inducer. Preferably said nucleic acid sequence is essentially not expressed in absence of said inducer.
A nucleic acid sequence of interest is operably linked to an inducible nucleic acid expression system when said inducible nucleic acid expression system is capable of expressing said nucleic acid sequence of interest. Preferably said nucleic acid sequence of interest is under control of a tetO-containing promoter. Expression of said nucleic acid of interest is at least in part inhibited in absence of an inducer, since rtTA and/or sc rtTA does not activate tetO-driven expression when an inducer is absent. In the presence of an inducer, rtTA and/or sc rtTA are able to activate tetO-driven expression of said nucleic acid of interest.
An rtTA or sc rtTA nucleic acid sequence according to the invention is defined as an rtTA or sc rtTA nucleic acid sequence derived from an rtTA or sc rtTA sequence (Urlinger et al. 2000; Das et al. 2004; Krueger et al. 2003), which rtTA or sc rtTA sequence has been provided with at least one mutation according to the present invention. Preferably at least one mutation according to the invention is introduced into the rtTA sequence depicted in
An rtTA nucleic acid and/or a sc rtTA nucleic acid is provided with a mutation of the present invention in a variety of ways. It is for instance possible to artificially introduce at least one mutation according to the present invention in an rtTA or sc rtTA nucleic acid, for instance via site directed mutagenesis. Various methods for artificially introducing a specific mutation are known in the art and do not require further explanation here. Once a mutation or a combination of mutations according to the invention is introduced, an rtTA or sc rtTA nucleic acid of the invention is preferably further amplified. Amplified rtTA or sc rtTA comprising a mutation according to the invention is thus also herewith provided. It is clear that it is no longer necessary to artificially introduce a mutation according to the invention once an rtTA or sc rtTA nucleic acid sequence of the invention is available, since a mutation of the invention is retained during amplification.
It is also possible to select an rtTA and/or sc rtTA with at least one mutation according to the invention from a collection of rtTA/sc rtTA nucleic acids. For instance, non-specific mutations are introduced into a collection of rtTA/sc rtTA nucleic acids, and a nucleic acid molecule comprising at least one mutation according to the invention is selected (optionally after amplification). In a preferred embodiment rtTA or sc rtTA nucleic acid of the invention is selected from a collection of amplified rtTA or sc rtTA via an evolution and selection method. Since a mutation of the invention provides at least one advantage to an rtTA/sc rtTA nucleic acid, it is possible to select a nucleic acid with a mutation of the invention on the basis of such advantage. For instance, a mutation of the invention resulting in enhanced sensitivity for dox is selected using very small amounts of dox. An inducible gene expression system is incubated with a very small amount of dox, and sensitive systems are selected. As another example, a mutation of the invention resulting in diminished basal activity is selected by selecting an inducible nucleic acid expression system with very low—if at all—activity in the absence of an inducer.
In one embodiment, forced evolution is used in order to generate and select an rtTA and/or sc rtTA nucleic acid with at least one mutation according to the invention. In such method, amplification of rtTA or sc rtTA is performed with a method which involves the introduction of mismatches. This is preferably performed using a genome of a virus comprising RNA, because the error-prone nature of its replication machinery (e.g. the reverse transcriptase (RT) enzyme or RNA polymerase enzyme) allows for the generation of modified nucleic acid sequences. This way, altered rtTA/sc rtTA nucleic acid molecules are produced. If such altered nucleic acid sequence comprises a mutation according to the invention, said nucleic acid will have an advantage over nucleic acid sequences without a mutation of the invention. As a result, nucleic acid molecules comprising at least one mutation according to the invention will outgrow nucleic acid molecules without a mutation of the invention. As a result, an rtTA and/or sc rtTA nucleic acid comprising at least one mutation according to the invention is easily selected.
In one preferred embodiment a forced evolution method is used with help of a Human Immunodeficiency Virus-1 (HIV-1) genome, which forced evolution method is described in WO 01/20013 page 21, lines 5-28. The description of the forced evolution methods of WO 01/20013 is incorporated herein by reference.
As used herein, an rtTA or sc rtTA variant is represented by the term “X[number]Y”, wherein X represents the kind of amino acid residue present in a currently used rtTA activator, [number] represents the position of said amino acid residue in said rtTA, and Y represents the amino acid residue that is currently present at said position in said variant. For instance, V9I means a variant which comprises at rtTA amino acid position 9 an isoleucine residue instead of a valine residue. Variants comprising multiple mutations are represented by multiple X[number]Y indications. Hence, variant V9I F67S R171K F86Y means a variant which comprises at rtTA amino acid position 9 an isoleucine instead of a valine and which comprises at rtTA amino acid position 67 a serine instead of a phenylalanine and which comprises at rtTA amino acid position 171 a lysine instead of an arginine and which comprises at rtTA amino acid position 86 a tyrosine instead of a phenylalanine.
Which mutation according to the invention, or which combination of mutations according to the invention, is used in a specific application is for instance dependent on the kind of advantage(s) that is desired. For instance, for inducible in vivo transgene expression in a brain, a sensitive rtTA and/or sc rtTA nucleic acid is particularly desired, since only a small amount of inducer is capable of passing the blood brain barrier. For such application, an rtTA/sc rtTA nucleic acid with a mutation according to the invention at least resulting in improved sensitivity is preferred. In that case, variant V9I F67S G138D F86Y, V9I F67S G138D F86Y A209T, V9I F67S E157K F86Y, V9I F67S E157K F86Y A209T, V9I F67S R171K F86Y, V9I F67S R171K F86Y A209T, E37Q F67S F86Y, E37Q F67S F86Y A209T, V9I C68R G138D F86Y, V9I C68R G138D F86Y A209T, V9I G19M G138D F86Y, V9I G19M G138D F86Y A209T, V9I E37Q G138D F86Y, V9I E37Q G138D F86Y A209T, V9I G19M F67S G138D F86Y, V9I G19M F67S G138D F86Y A209T, V9I S12G F67S G138D F86Y, V9I S12G F67S G138D F86Y A209T, V9I F67S C68R G138D F86Y and/or V9I F67S C68R G138D F86Y A209T is preferred because these variants have more than 100-fold doxycyclin sensitivity as compared to rtTA, with no or low basal activity in the absence of inducer (as indicated in
Furthermore, an rtTA or sc rtTA variant comprising an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine valine or tyrosine residue at rtTA amino acid position 19 has an improved transcriptional activity as compared to currently known rtTA. Said variants have an increased transcriptional activity at a low doxycycline concentration (between 10 and 100 ng/ml) and/or an increased transcriptional activity at a high doxycycline concentration (between 100 and 1000 ng/ml) as compared to currently known rtTA. One embodiment of the invention therefore provides an isolated, synthetic or recombinant amino acid sequence comprising an rtTA sequence and/or a sc rtTA sequence, which rtTA sequence and/or sc rtTA sequence comprises an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine valine or tyrosine at rtTA amino acid position 19. An isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an rtTA sequence and/or a sc rtTA sequence, which rtTA sequence and/or sc rtTA sequence comprises an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine valine or tyrosine at rtTA amino acid position 19 is also herewith provided, as well as a use of said nucleic acid sequence in a method according to the invention for inducible expressing a nucleic acid sequence of interest.
An rtTA or sc rtTA variant comprising a cysteine, methionine, glutamine, arginine or threonine residue at rtTA amino acid position 37 has an improved transcriptional activity as compared to currently known rtTA. Said variants have an increased transcriptional activity at a low doxycycline concentration (between 10 and 100 ng/ml) and/or an increased transcriptional activity at a high doxycycline concentration (between 100 and 1000 ng/ml) as compared to currently known rtTA. One embodiment of the invention therefore provides an isolated, synthetic or recombinant amino acid sequence comprising an rtTA sequence and/or a sc rtTA sequence, which rtTA sequence and/or sc rtTA sequence comprises a cysteine, methionine, glutamine, arginine or threonine residue at rtTA amino acid position 37. An isolated, synthetic or recombinant nucleic acid sequence comprising a sequence encoding an rtTA sequence and/or a sc rtTA sequence, which rtTA sequence and/or sc rtTA sequence comprises a cysteine, methionine, glutamine, arginine or threonine residue at rtTA amino acid position 37 is also herewith provided, as well as a use of said nucleic acid sequence in a method according to the invention for inducible expressing a nucleic acid sequence of interest.
If an rtTA and/or sc rtTA nucleic acid of the invention is used for vaccination purposes involving controlled expression of a (pathogen-derived) nucleic acid sequence of interest, it is important that basal activity is minimal. Expression of a pathogenic nucleic acid of interest in absence of an inducer is undesired because it would result in continuous presence of said pathogenic nucleic acid of interest. In that case an organism would be challenged too much with pathogenic nucleic acid, which could for instance result in disease and/or tolerance of the immune system for said pathogenic nucleic acid of interest. If tolerance is induced, protection against a subsequent challenge with said pathogen is diminished. At least in part avoiding basal activity is particularly important if the replication of a viable pathogen is inducibly controlled by an rtTA and/or sc rtTA system. Continuous replication of said pathogen involves the risk of spreading and outgrowth of too many pathogenic organisms, resulting in disease. For vaccination purposes, an rtTA and/or sc rtTA variant of the invention with a very low basal activity—if any—is therefore preferred. In such case, variant F67S F86Y, F67S F86Y A209T, G138D F86Y, G138D F86Y A209T, E157K F86Y, E157K F86Y A209T, R171K F86Y, R171K F86Y A209T, V9I G138D F86Y, V9I G138D F86Y A209T, V9I E157K F86Y, V9I E157K F86Y A209T, V9I R171K F86Y, V9I R171K F86Y A209T, F177L F86Y, F177L F86Y A209T, F67S F177L F86Y, F67S F177L F86Y A209T, C195S F86Y, C195S F86Y A209T, G138S F86Y, G138S F86Y A209T, C68R F86Y, C68R F86Y A209T, F67S G138D F86Y, F67S G138D F86Y A209T, F67S E157K F86Y, F67S E157K F86Y A209T, F67S R171K F86Y, F67S R171K F86Y A209T, V9I F67S R171K F86Y, V9I F67S R171K F86Y A209T, S12G F67S F86Y, S12G F67S F86Y A209T, G19M F67S F86Y and/or G19M F67S F86Y A209T is preferably used. These variants have a very low basal activity of 0.1 percent or less. A V9I F67S R171K F86Y and/or V9I F67S R171K F86Y A209T rtTA variant is most preferably used. These variants have a very low basal activity and are very sensitive since they have a more than 100-fold doxycyclin sensitivity as compared to rtTA (indicated in
The invention furthermore provides rtTA and sc rtTA variants with altered inducer-specificities. rtTA and sc rtTA variants are provided that are inducible by antibiotics other than doxycycline. Hence, although other dox-like compounds such as tetracycline (Tc) and minocycline (Mc) do not effectively activate wild type rtTA, the invention provides variants that have become inducible by at least one of these antibiotics. This provides amongst other things the advantage that tetracycline is suitable as inducer, which is cheaper than doxycycline. Furthermore, rtTA and sc rtTA variants according to the invention that are inducible by antibiotics other than doxycycline are suitable for the development of rtTA and/or sc rtTA variants with an altered specificity which are inducible by at least one antibiotic other than doxycycline but not by doxycycline itself.
The invention therefore provides an isolated, synthetic or recombinant nucleic acid sequence comprising an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutation or a combination of mutations as depicted in
Preferably, mutant F67S V9I G138D F86Y A209T, C68R V9I G138D F86Y A209T, G19M V9I G138D F86Y A209T, E37Q V9I G138D F86Y A209T, G19M F67S V9I G138D F86Y A209T, S12G F67S V9I G138D F86Y A209T and/or C68R F67S V9I G138D F86Y A209T is used for tetracycline-inducible expression of a nucleic acid sequence of interest since these mutants are particularly sensitive for tetracycline, meaning that a small amount of tetracycline is sufficient for inducing gene expression. Most preferably, mutant F67S V9I G138D F86Y A209T, C68R V91 G138D F86Y A209T and/or S12G F67S V9I G138D F86Y A209T is used for tetracycline-inducible expression of a nucleic acid sequence of interest since these mutants are very sensitive for tetracycline and show low background activity in the absence of any effector.
In a further preferred embodiment, mutant V9I G138D F86Y A209T, F67S V9I G138D F86Y A209T, F67S V9I E157K F86Y A209T, F67S V9I R171K F86Y A209T, F67S E37Q F86Y A209T, C68R V9I G138D F86Y A209T, G19M V9I G138D F86Y A209T, E37Q V9I G138D F86Y A209T, G19M F67S V9I G138D F86Y A209T, S12G F67S V9I G138D F86Y A209T and/or C68R F67S V9I G138D F86Y A209T is used for minocycline-inducible expression of a nucleic acid sequence of interest since these mutants are particularly sensitive for minocycline, meaning that a small amount of minocycline is sufficient for inducing gene expression. Most preferably, mutant F67S V9I G138D F86Y A209T, F67S E37Q F86Y A209T, C68R V9I G138D F86Y A209T and/or S12G F67S V9I G138D F86Y A209T is used for minocycline-inducible expression of a nucleic acid sequence of interest since these mutants are very sensitive for minocycline and show low background activity in the absence of any effector.
The invention further provides rtTA and sc rtTA variants that are genetically stable. Currently used inducible Tet-on systems are at risk of converting into a system constitutively expressing a nucleic acid sequence of interest. This is preferably avoided, for instance (amongst other things) when replication of a pathogen is controlled by a Tet-on system. Constitutive replication of said pathogen would result in the presence of too many pathogens, involving the risk of disease and/or tolerance.
According to the present invention, the genetic stability of an inducible gene expression system comprising rtTA and/or sc rtTA nucleic acid is improved by altering a codon at rtTA amino acid position 19, 37 and/or 56 (and/or the corresponding codons in sc rtTA) such that loss of inducer dependency is at least in part prevented. Reduction and/or loss of inducer-dependence of currently used rtTA and sc rtTA result from at least one mutation at rtTA amino acid position 19, 37 and/or 56, such as for instance a G19E, E37K, E37A, E37S and/or a P56S mutation. According to the invention, replacement of the glycine residue at rtTA amino acid position 19 by a glutamate residue results in at least partial loss of inducer-dependence of rtTA/sc rtTA. Moreover, replacement of the glutamate residue at rtTA amino acid position 37 by a lysine, alanine or serine residue results in at least partial loss of inducer-dependence of rtTA/sc rtTA. Moreover, replacement of the proline residue at rtTA amino acid position 56 by serine, tyrosine, cysteine, histidine, asparagine, alanine or glycine results in at least partial loss of inducer-dependence of rtTA/sc rtTA. The invention therefore provides modified rtTA and/or sc rtTA nucleic acids wherein spontaneous mutations that would result in at least partial loss of inducer-dependence are less likely to occur as compared to currently used rtTA/sc rtTA. Such variants are obtained as described in the following paragraph.
In currently used rtTA, a G19E mutation requires only one nucleotide change in codon 19, namely the codon change GGA to GAA. The G-to-A transition is the most frequent error during reverse transcription of RNA. According to the present invention, reduction and/or loss of inducer-dependency of rtTA and/or sc rtTA is at least in part prevented by using an altered codon at position 19 that is more difficultly converted into a glutamate codon. This is preferably performed by using a codon at position 19 that differs in at least two nucleotides from a glutamate codon. If such codon is used, an undesired G19E mutation would require a much more difficult two-hit mutation. Hence, when an rtTA and/or sc rtTA nucleic acid is used with a codon at rtTA position 19 which differs in at least two nucleotides from a glutamate codon, an undesired G19E mutation is less likely to evolve as compared to currently used Tet-on systems. Reduction and/or loss of inducer-dependence is therefore at least in part prevented. The invention therefore provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon.
In one embodiment an alternative glycine codon at position 19 is used which alternative glycine codon differs in at least two nucleotides from a glutamate codon. In this embodiment, the alternative glycine codon GGU or GGC is used (instead of GGA which is present in currently used rtTA). A G19E mutation is much more difficult in this embodiment because it requires a GGU to GAA, a GGU to GAG, a GGC to GAA or a GGC to GAG change. Hence, in all those cases a two-hit mutation would be required. Since this is less likely to occur, an rtTA and/or sc rtTA with an alternative glycine codon according to this embodiment is less likely to lose its inducer-dependence. When an alternative glycine codon according to this embodiment is used, the resulting amino acid residue of the rtTA or sc rtTA activator at rtTA position 19 is the same as the activator encoded by currently used rtTA and sc rtTA nucleic acid. One preferred embodiment therefore provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a glycine codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon.
In a more preferred embodiment, an rtTA or sc rtTA nucleic acid is used which comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon. A nucleic acid according to this embodiment is not only genetically more stable, but—except for the G19W variant—is also more sensitive for doxycycline. One embodiment therefore provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon. Suitable codons at rtTA amino acid position 19 which differ in at least two nucleotides from a glutamate codon are codon UUN (with N corresponding to G, A, U, or C (coding for Phenylalanine or Leucine), UCN (Serine), UAY (with Y corresponding to U or C; Tyrosine), UGU (Cysteine), UGC (Cysteine), UGG (Tryptophan), CUN (Leucine), CAY (Histidine), CGN (Arginine), AUN (Isoleucine or Methionine), ACN (Threonine), AAY (Asparagine), AGN (Serine or Arginine), GUY (Valine) and GCY (Alanine).
In another preferred embodiment, a method according to the invention is provided wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a cysteine, phenylalanine, isoleucine, leucine, arginine, serine or threonine codon at rtTA amino acid position 19 which differs in three nucleotides from a glutamate codon. Such variant is in particular genetically stable because three mutations would be required in order to generate a G19E variant. Suitable codons at rtTA amino acid position 19 which differ in at least three nucleotides from a glutamate codon are codon UUY (with Y corresponding to U or C; coding for Phenylalanine), UCY (Serine), UGY (Cysteine), CUY (Leucine), CGY (Arginine), AUY (Isoleucine), ACY (Threonine) and AGY (Serine).
An rtTA or sc rtTA nucleic acid comprising a codon at rtTA amino acid position 37 which differs in at least two nucleotides from an alanine, a lysine and a serine codon is also provided. If such variant is used, spontaneous E37K, E37A and E37S mutation is less likely to occur as compared to currently used rtTA/sc rtTA because that would require a much more difficult two-hit mutation. As a consequence, loss of inducer dependency is at least in part avoided. The invention therefore provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon. Suitable codons at rtTA amino acid position 37 which differ in at least two nucleotides from an alanine, a lysine or a serine codon are codon CUN (coding for leucine, N stands for U, C, A or G), CAU, CAC (both CAU and CAC coding for histidine), CGA and CGG (both CGA and CGG coding for arginine). A rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprising codon CUN, CAU, CAC, CGA or CGG at rtTA amino acid position 37 is therefore preferably provided.
One preferred embodiment provides a method according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon and a codon at rtTA amino acid position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon. Such variant is particularly genetically stable, since spontaneous G19E, E37K, E37A and E37S mutations are at least in part prevented.
An rtTA and/or sc rtTA nucleic acid comprising an altered codon at rtTA amino acid position 56 also provides enhanced stability. It has been found that in the absence of doxycycline, rtTA variants are at risk of evolving to variants that have an altered amino acid residue at rtTA amino acid position 56 and that are no longer dependent on doxycycline. The invention therefore provides an isolated, recombinant or synthetic nucleic acid sequence comprising an rtTA and/or sc rtTA encoding nucleic acid sequence which comprises a codon at rtTA amino acid position 56 which differs in at least one nucleotide, preferably a transversion, from a codon that mediates transcriptional activity in the absence of an inducer. Most of said doxycycline-independent rtTA variants contain either a serine, tyrosine, cysteine, histidine, asparagine, alanine or glycine residue at position 56 instead of a proline. In order to at least in part avoid the development of such variant, an rtTA and/or sc rtTA encoding nucleic acid is preferably provided which comprises a CAA or CAG codon encoding glutamine or a AAA or AAG codon encoding lysine at rtTA amino acid position 56.
A transversion is defined herein as a substitution of a purine into a pyrimidine, or a substitution of a pyrimidine into a purine. A transversion is less likely to occur during natural evolution as compared to a substitution of a purine into another purine, or a substitution of a pyrimidine into another pyrimidine. Therefore, an rtTA and/or sc rtTA encoding nucleic acid sequence which comprises a codon at rtTA amino acid position 56 which differs in at least one transversion from a codon encoding a serine, tyrosine, cysteine, histidine, asparagine, alanine or glycine residue is genetically more stable as compared to current rtTA and sc rtTA.
According to the present invention, an rtTA or sc rtTA nucleic acid with at least one mutated codon at rtTA amino acid position 9, 19, 37, 56, 67, 68, 138, 157, 171, 177, and/or 195 comprises at least one improved characteristic as compared to currently available Tet-on systems. Preferably, a method according to the invention is provided wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding threonine, histidine, leucine, arginine, cysteine, methionine or glutamine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine. Any of these mutations, or any combination of them, is preferred since they particularly improve at least one property of an inducible nucleic acid expression system.
In order to improve rtTA and/or sc rtTA, it is sufficient to introduce one mutation according to the invention into an rtTA and/or sc rtTA encoding nucleic acid sequence. Preferably however at least two mutations according to the invention are introduced, since a combination of at least two mutations according to the invention further improves at least one property of rtTA and/or sc rtTA. In a more preferred embodiment an rtTA nucleic acid and/or a sc rtTA nucleic acid comprises at least three mutations according to the present invention. Most preferably, an rtTA nucleic acid and/or a sc rtTA nucleic acid comprises at least four mutations according to the present invention.
In
A mutation according to the present invention is furthermore suitable for improving at least one characteristic of alternative rtTA and/or TetR derived molecules, besides rtTA and sc rtTA. Such alternative rtTA and/or TetR derived molecule for instance comprises an alternative transcriptional activation domain (see for example [Akagi et al. 2001] and [Kamper et al. 2002]), or a transcriptional silencer in which the activation domain has been replaced by a transcriptional repressor domain (tTS, see for example [Deuschle et al. 1995]), or the tTA transcriptional activator, which is active in the absence of an effector and repressed by an effector (Gossen and Bujard, 1992).
Any inducible nucleic acid expression system comprising an rtTA and/or sc rtTA nucleic acid sequence according to the present invention and/or an alternative molecule derived thereof is suitable for inducibly expressing a nucleic acid sequence of interest. In vivo as well as ex vivo applications are possible. In one embodiment a prokaryotic nucleic acid expression system is used. Preferably said nucleic acid of interest is expressed in a eukaryotic expression system, because an rtTA and/or sc rtTA sequence comprising a VP16 activation domain of herpes simplex virus is particularly suitable for regulating nucleic acid expression from tetO-containing promoters in eukaryotic cells. An rtTA and/or sc rtTA nucleic acid sequence according to the present invention, and alternative molecules derived thereof, are suitable for use in a lower eukaryotic expression system. Moreover, an rtTA and/or sc rtTA nucleic acid sequence according to the present invention and alternative molecules derived thereof are suitable for use in a higher eukaryotic expression system. In one embodiment said nucleic acid of interest and/or an alternative molecule derived thereof is expressed in a mammalian cell.
In principle, any nucleic acid sequence of interest is inducibly expressed by a nucleic acid expression system according to the present invention. For instance, suitable applications for an inducible gene expression system according to the present invention are the production of protein pharmaceuticals, in vivo expression of therapeutic proteins and production of transgenic animals wherein a (pathogenic) gene of interest is inducibly expressed, to name just a few. In one embodiment at least one viral sequence essential for replication of a virus or replicon is inducibly expressed by a nucleic acid expression system of the invention. This is particularly suitable for vaccination purposes in order to provide at least partial protection to a viral pathogen, wherein it is important that said virus or replicon replicates in order to obtain an efficacious immune response, but wherein it is also important that said replication does not go beyond the level required for said immune response. A replicon is defined as a nucleic acid molecule capable of replication in a suitable environment, such as a permissive cell, because it has all the necessary elements for replication in such an environment. We call it a replicon, because it will not always be directly derived from the nucleotide sequences of the original pathogen.
By placing at least one viral sequence essential for replication of a virus or replicon under control of an rtTA and/or sc rtTA nucleic acid sequence of the invention, said virus or replicon replicates in a controlled manner. The amount of replication necessary for eliciting a good immune response without any replication beyond that level is thus regulated by regulating the amount of inducer that is administered to an inducible nucleic acid expression system according to the present invention. In order to prevent leakage, it is preferred to have a combination of essential genes under such control and it is even more preferred to have at least two different repressor/activator combinations in control of at least one, but preferably more than one gene essential for replication. In most (viral) pathogens a number of genes is essential for replication, but most of them also have a sort of “master switch”, usually an early gene, usually transactivating other genes. A first candidate to put under direct control of a repressor/activator is of course such a master switch, which then indirectly provides control over other essential genes for replication. Still it is even then preferred to put at least one other essential gene under control of an inducible repressor/activator.
In one embodiment at least part of a HIV genome essential for replication is inducibly expressed under control of an rtTA and/or sc rtTA nucleic acid sequence of the invention. This is for instance suitable for improved AIDS prophylaxis as compared to currently known methods. In this embodiment a master switch is not required since an HIV genome is under control of a single transcription unit.
A nucleic acid sequence comprising an rtTA nucleic acid sequence and/or a sc rtTA nucleic acid sequence, said rtTA nucleic acid sequence and/or sc rtTA nucleic acid sequence comprising at least one mutation according to the present invention, finds utility in a wide variety of applications. Said nucleic acid sequence is particularly suitable for use in an inducible nucleic acid sequence expression system. The invention thus provides an isolated, synthetic or recombinant nucleic acid sequence comprising an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence, which rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a mutated codon at rtTA amino acid position 9, and/or 19, and/or 37, and/or 56, and/or 67, and/or 68, and/or 138, and/or 157, and/or 171, and/or 177, and/or 195. In one embodiment said nucleic acid sequence further comprises a mutated codon at rtTA amino acid position 12, and/or 86, and/or 209. A nucleic acid sequence comprising such combination of mutations is also improved as compared to currently known rtTA.
In a preferred embodiment a nucleic acid sequence of the invention with an improved genetic stability as compared to currently used Tet-on systems is provided. This is particularly desired in applications involving multiple rounds of amplification of a nucleic acid sequence according to the invention, for instance during controlled replication of a viral pathogen or replicon. As explained above, genetic stability is improved by designing an rtTA and/or sc rtTA nucleic acid sequence with a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon, with a codon at rtTA position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or with a codon at rtTA position 56 encoding lysine or glutamine. Further provided is therefore an isolated, synthetic or recombinant nucleic acid sequence according to the invention wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon, a codon at rtTA position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon, and/or a codon at rtTA position 56 encoding lysine or glutamine. In one embodiment said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a glycine codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon, so that the resulting amino acid residue of the rtTA or sc rtTA activator at rtTA position 19 is the same as the activator encoded by currently used rtTA and sc rtTA nucleic acid.
Preferably an rtTA or sc rtTA nucleic acid is used which comprises an alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophan or tyrosine codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon. A nucleic acid according to this embodiment is not only genetically more stable as compared to currently used Tet-on systems, but—except for the G19W variant—is also more sensitive for doxycycline. One preferred embodiment provides an isolated, synthetic or recombinant nucleic acid sequence according to the invention, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a leucine, a histidine or an arginine codon at rtTA amino acid position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon.
A further preferred embodiment provides an isolated, synthetic or recombinant nucleic acid sequence according to the invention, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 56 which differs in at least one nucleotide, preferably a transversion, from a codon that mediates transcriptional activity in the absence of inducer. Said codon at rtTA amino acid position 56 preferably encodes a glutamine or a lysine residue.
More preferably an isolated, synthetic or recombinant nucleic acid sequence according to the invention is provided which comprises a codon according to the invention at least two rtTA amino acid positions which are chosen from the group consisting of positions 19, 37 and 56. Preferably provided is therefore an isolated, synthetic or recombinant nucleic acid sequence according to the invention which comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon and a codon at rtTA position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon. Further provided is an isolated, synthetic or recombinant nucleic acid sequence according to the invention which comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon and a codon at rtTA position 56 encoding lysine or glutamine. Further provided is an isolated, synthetic or recombinant nucleic acid sequence according to the invention which comprises a codon at rtTA position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon and a codon at rtTA position 56 encoding lysine or glutamine.
An isolated, synthetic or recombinant nucleic acid sequence according to the invention most preferably comprises a codon according to the invention at rtTA amino acid position 19, 37 and 56. A preferred embodiment therefore provides an isolated, synthetic or recombinant nucleic acid sequence according to the invention which comprises a codon at rtTA amino acid position 19 which differs in at least two nucleotides from a glutamate codon, a codon at rtTA position 37 which differs in at least two nucleotides from an alanine, a lysine or a serine codon, and a codon at rtTA position 56 encoding lysine or glutamine.
As stated hereinbefore, a nucleic acid sequence according to the present invention preferably comprises an rtTA encoding nucleic acid sequence and/or a sc rtTA encoding nucleic acid sequence, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises a codon at rtTA amino acid position 9 encoding isoleucine, and/or a codon at rtTA amino acid position 19 encoding alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine, and/or a codon at rtTA amino acid position 37 encoding threonine, histidine, leucine, arginine, cysteine, methionine or glutamine, and/or a codon at rtTA amino acid position 56 encoding lysine or glutamine, and/or a codon at rtTA amino acid position 67 encoding serine, and/or a codon at rtTA amino acid position 68 encoding arginine, and/or a codon at rtTA amino acid position 86 encoding tyrosine, and/or a codon at rtTA amino acid position 138 encoding aspartate or serine, and/or a codon at rtTA amino acid position 157 encoding lysine, and/or a codon at rtTA amino acid position 171 encoding lysine, and/or a codon at rtTA amino acid position 177 encoding leucine, and/or a codon at rtTA amino acid position 195 encoding serine, and/or a codon at rtTA amino acid position 209 encoding threonine. These mutations are preferred since each of them particularly improves at least one property of an inducible nucleic acid expression system. Hence, either one of these mutations or any combination thereof is preferably present in a nucleic acid sequence of the invention.
Further provided is an isolated, synthetic or recombinant nucleic acid sequence according to the invention, wherein said rtTA encoding nucleic acid sequence and/or single chain rtTA encoding nucleic acid sequence comprises at least one variant as depicted in
A nucleic acid sequence according to the invention preferably comprises an rtTA encoding nucleic acid sequence and/or a single chain rtTA encoding nucleic acid sequence which comprises at least one mutation as compared to a rtTA or sc rtTA encoding nucleic acid sequence published in (Gossen et al. 1995), (Urlinger et al. 2000) and (Krueger et al. 2003) and/or depicted in
The invention furthermore provides an isolated, synthetic or recombinant amino acid sequence encoded by a nucleic acid sequence according to the invention. Said amino acid sequence preferably comprises an rtTA sequence and/or a single chain rtTA sequence, which rtTA sequence and/or single chain rtTA sequence comprises an isoleucine at position 9, and/or an alanine, cysteine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, arginine, serine, threonine, valine, aspartate, glutamine, tryptophan or tyrosine at position 19, and/or a threonine, histidine, leucine, arginine, cysteine, methionine or glutamine at position 37, and/or a lysine or glutamine at position 56, and/or a serine at position 67, and/or an arginine at position 68, and/or a tyrosine at position 86, and/or an aspartate or serine at position 138, and/or a lysine at position 157, and/or a lysine at position 171, and/or a leucine at position 177, and/or a serine at position 195, and/or a threonine at position 209. Each of these mutations particularly confer at least one improved property to an rtTA and/or sc rtTA activator.
As explained above, a nucleic acid sequence of the invention and/or an amino acid sequence encoded by a nucleic acid sequence of the invention is particularly suitable for inducibly expressing a nucleic acid sequence of interest. Use of an isolated, synthetic or recombinant nucleic acid sequence and/or amino acid sequence according to the invention for inducible expression of a nucleic acid sequence of interest is therefore also herewith provided.
Said amino acid sequence preferably comprises at least one of the above mentioned mutations.
Further provided is a vector comprising a nucleic acid sequence according to the invention. Such vector is suitable for a variety of applications. For instance, a vector of the invention comprising a therapeutically beneficial nucleic acid sequence is suitable for therapeutic applications. Administration of such vector to an individual in need thereof results in expression of said therapeutic nucleic acid sequence in vivo. Of course, said vector also finds utility in applications involving in vitro expression of a nucleic acid sequence of interest. Methods for constructing a vector with a particular nucleic acid sequence are well known in the art. Non-limiting examples of vectors suitable for generating a vector of the invention are retroviral and lentiviral vectors.
An inducible viral replicon, comprising a nucleic acid sequence according to the invention and at least one viral sequence which is essential for replication under direct or indirect control of said nucleic acid sequence is also herewith provided. As explained before, a replicon is defined as a nucleic acid molecule capable of replication in a suitable environment, such as a permissive cell, because it has all the necessary elements for replication in such an environment. We call it a replicon, because it will not always be directly derived from the nucleotide sequences of the original pathogen, for instance in the case of single stranded DNA viruses, RNA viruses, etc. Typically, in order to manipulate nucleic acids, double stranded forms are necessary, typically double stranded DNA forms. Therefore preferred replicons will be double stranded DNA nucleic acids in at least one stage of their life cycle.
A replicon is also intended to reflect that the actual pathogen, or its attenuated live vaccine relative, usually comprises more than just nucleic acid. The nucleic acid is typically packaged into a (viral) particle. Therefore a replicon according to the invention preferably also encodes a functional packaging signal, allowing for the nucleic acid in its wild-type-like form (RNA in the case of a retrovirus, etc.) to be packed into a viral particle. In order for the replicon to be able to replicate in a host, it is preferred that said replicon also carries the structural genes for the proteins of the envelope and/or capsid, be it in wild-type format or in a somewhat different format (reduced or enhanced target binding, etc.). In order to enhance inducer-dependency of a viral replicon according to the invention and/or to at least in part prevent loss of inducer-dependency, an inducible viral replicon according to the invention preferably comprises all viral sequences which are essential for replication under direct or indirect control of said nucleic acid sequence
A viral replicon of the invention is preferably derived from any virus comprising a stage wherein at least part of its genome is present in the form of DNA, such that the Tet-on machinery is capable of regulating expression of a nucleic acid of interest. Such viruses for instance comprise DNA viruses and retroviruses. In one embodiment at least part of said viral sequences in said inducible replicon is RNA.
In one preferred embodiment a replicon according to the invention is derived from a human immunodeficiency virus. A replicon according to the invention is now further exemplified by the preferred embodiments relating to a replicon derived from HIV. However, the invention is also applicable to replicons derived from other pathogens.
Typically a replicon of the invention derived from HIV would be an infectious double stranded DNA clone of an HIV strain. Preferably said HIV strain is already an attenuated strain, or is made into an attenuated strain by introducing mutations, such as functional deletions, e.g. those described herein. Any repressor/activator elements that are inducible are in principle applicable in the present invention. In the case of double or more inducible controls, leakage of a single repressor/activator becomes less important, although essentially no leakage is still highly preferred. As a safety valve, it would be advantageous to provide the replicon with a suicide gene that can be activated when unwanted effects occur such as replication beyond what is necessary for an immune response or rescue by wild type virus, etc. Such a suicide gene is e.g. HSV-tk, which can be induced by adding gancyclovir or a functional equivalent thereof. Upon induction said suicide gene will kill the infected cell, and thereby inhibit further replication and infection of other cells. Thus in yet another preferred embodiment the invention provides a replicon according to the invention which further comprises a suicide gene.
In order to attenuate an HIV replicon and/or the resulting virus it is preferred that the replicon is provided with a functional deletion of the TAR-element. Thus in yet another preferred embodiment the invention provides a replicon according to the invention, which further comprises an inactivated TAR element.
In order to attenuate an HIV replicon according to the invention it is preferred to functionally delete the Tat element. Thus the invention also provides a replicon according to the invention, which further comprises an inactivated Tat element. Preferably both elements mentioned above are functionally deleted. Functional deletion means that at least their function in the replication of the replicon is at least partially inhibited. Essential genes for replication typically should not be completely dysfunctional.
Proteins necessary for removing repression or initiating activation elements which are present upstream of the essential genes to be put under control are preferably encoded by a replicon according to the invention and are preferably inserted in a non-essential gene. Thus the invention also provides in a preferred embodiment a replicon according the invention wherein at least one functional part of said inducible repressor and/or activator, preferably an rtTA and/or sc rtTA nucleic acid sequence according to the present invention, is inserted into the nef gene. The functional part in this case of course refers to any proteinaceous substance capable of activating the element in control of the essential gene. Preferably space is created for the sequence encoding said proteinaceous substance. Thus the invention also provides a replicon according to the invention in which at least part of the nef gene is deleted to create space for insertion.
To further attenuate a replicon according to the invention further elements of the wild-type virus can be functionally deleted. Thus the invention further provides a replicon according to the invention, in which at least one NF-kB element has been deleted. It is preferred that the motif to be activated is a tetO motif, preferably present in an LTR. Thus the invention also provides a replicon according to the invention, which comprises at least one tetO motif in at least one functional LTR.
It is preferred to have more than one element before an essential gene. Thus the invention also provides a replicon which comprises at least 2, 4, 6, or 8 such elements in at least one functional LTR.
At least one LTR is preferably modified in order to at least in part avoid reversion to wild type virus.
The invention further provides methods using the replicons to produce dependent viruses, meaning viruses needing an inducing agent in order to be able to replicate. Thus the invention provides a method for producing a virus dependent on an inducing agent for replication, comprising providing a permissive cell with a replicon according to the invention, culturing said cell in the presence of said inducing agent and harvesting said dependent virus from said culture. Again such methods are preferably applied to HIV. Thus the invention provides a method in which said dependent virus is a human immunodeficiency virus, preferably an attenuated virus.
In one embodiment the inducing agent is doxycyclin or a functional analog thereof. In another embodiment however said inducing agent comprises an antibiotic other than doxycyclin, preferably tetracyclin and/or minocyclin. As stated before, if tetracyclin and/or minocyclin-dependency is desired, a replicon according to the invention is preferred which comprises an rtTA and/or sc rtTA encoding nucleic acid sequence comprising
a mutation or a combination of mutations as depicted in
More preferably, a replicon according to the invention comprising mutations F67S V9I G138D F86Y A209T, C68R V9I G138D F86Y A209T, G19M V9I G138D F86Y A209T, E37Q V9I G138D F86Y A209T, G19M F67S V9I G138D F86Y A209T, S12G F67S V9I G138D F86Y A209T and/or C68R F67S V9I G138D F86Y A209T is used for tetracycline-inducible expression of a nucleic acid sequence of interest since these replicons are particularly sensitive for tetracycline, meaning that a small amount of tetracycline is sufficient for inducing gene expression. Most preferably, a replicon according to the invention comprising mutation F67S V9I G138D F86Y A209T, C68R V9I G138D F86Y A209T and/or S12G F67S V9I G138D F86Y A209T is used for tetracycline-inducible expression of a nucleic acid sequence of interest since these replicons are very sensitive for tetracycline and show low background activity in the absence of effector.
In a further preferred embodiment, a replicon according to the invention comprising mutations V9I G138D F86Y A209T, F67S V9I G138D F86Y A209T, F67S V9I E157K F86Y A209T, F67S V9I R171K F86Y A209T, F67S E37Q F86Y A209T, C68R V9I G138D F86Y A209T, G19M V9I G138D F86Y A209T, E37Q V9I G138D F86Y A209T, G19M F67S V9I G138D F86Y A209T, S12G F67S V9I G138D F86Y A209T and/or C68R F67S V9I G138D F86Y A209T is used for minocycline-inducible expression of a nucleic acid sequence of interest since these replicons are particularly sensitive for minocycline, meaning that a small amount of minocycline is sufficient for inducing gene expression. Most preferably, a replicon according to the invention comprising mutations F67S V9I G138D F86Y A209T, F67S E37Q F86Y A209T, C68R V9I G138D F86Y A209T and/or S12G F67S V9I G138D F86Y A209T is used for minocycline-inducible expression of a nucleic acid sequence of interest since these replicons are very sensitive for minocycline and show low background activity in the absence of effector.
Also part of the present invention are viruses produced by said methods or which can be produced by said methods. Thus the invention also provides a virus dependent on an inducing agent for replication obtainable by a method according to the invention, preferably again a human immunodeficiency virus, again preferably attenuated. Methods for producing a replicon and/or virus according to the present invention are known in the art. For instance, non-limiting examples of methods for producing an inducible viral replicon derived from HIV, comprising an rtTA sequence and TetO elements, and uses thereof, are described in WO01/20013 page 9 line 13 to page 18 line 27. These methods and uses are incorporated herein by reference.
A replicon and/or virus according to the invention is particularly suitable for immunization and vaccination. Administration of a replicon and/or virus according to the invention to an individual allows for controlled replication of said replicon and/or virus within said individual, resulting in an immune response in said individual. The extent of replication of said replicon or virus and, as a result, the extent of elicited immune response is controlled by regulating the presence and/or amount of inducer. In one embodiment an immune response is elicited in an individual in order to provide said individual with at least partial protection against infection by the kind of virus from which said replicon or virus of the invention is derived. In another embodiment an immune response is elicited in a non-human animal in order to produce a binding compound (such as for instance antibodies and/or T cells) and/or a cell capable of producing such binding compound (such as a B cell). Said antibodies, T cells and/or B cells, or a functional part and/or a nucleic acid thereof, are preferably harvested for further use, for instance for the production of monoclonal antibodies.
Of course, various alternative methods and applications involving immunization and/or vaccination are known in the art. The use of a replicon and/or virus according to the invention in such methods and applications is also within the scope of the present invention.
The invention thus provides an immunogenic composition comprising a replicon according to the invention and/or a virus according to the invention. An immunogenic composition comprising a nucleic acid sequence according to the invention is also provided. An immunogenic composition of the invention preferably comprises a suitable adjuvant and/or carrier. Adjuvants and carriers are well known in the art. For instance, an Aluminum Salt Adjuvant and/or a saline solution is used.
In one embodiment said immunogenic composition further comprises an amount of inducing agent. This is however not necessary: an inducing agent can be administered at any time. In one preferred embodiment said immunogenic composition comprises a vaccine capable of eliciting full protection against the kind of virus from which said replicon or virus according to the invention is derived. This means that subsequent infection with the kind of virus from which said replicon or virus according to the invention is derived does essentially not result in disease.
An immunogenic composition or a vaccine may comprise a single dosage unit, but it may also comprise at least one inducing agent separately, or it may be made on the spot from a replicon and/or virus that are reconstituted with a liquid excipient such as saline, optionally together with an adjuvant and/or an inducing agent. Viral vaccines are well known in the field. General rules of thumb applicable to known vaccines will also apply to immunogenic compositions and vaccines of the present invention. Doses will be found through the normal dose finding studies performed during (pre)clinical trials, e.g. by simple titration of the amount of doxycycline as inducing agent. An immunogenic composition or vaccine may be sufficient on its own, but it may also be used in addition to other therapeutic and/or prophylactic compounds. The inducing agent may be needed over a longer period of time and can be provided separately.
Again a preferred immunogenic composition and/or vaccine of the invention is one for at least partial prophylaxis of infection with a human immunodeficiency virus.
The invention also provides a use of said immunogenic composition and/or vaccine in that it provides a method for at least partial prophylaxis and/or treatment of AIDS, comprising administering an immunogenic composition and/or vaccine according the invention to an individual and allowing for viral replication for a limited time by providing said inducing agent. Booster vaccinations are possible, preferably by simple readdition of the said inducing agent at later times.
The invention also provides a method for the controlled replication of a virus or a viral replicon comprising providing a permissive cell with a replicon or a virus according to the invention, culturing said cell in the presence of said inducing agent and manipulating the amount of inducing agent present.
As explained above, a replicon, virus and/or nucleic acid sequence according to the invention is suitable for eliciting an immune response against a virus of interest. Said immune response is capable of at least in part preventing subsequent infection, replication and/or spreading by said virus of interest. Moreover, an immune response of an individual that is already suffering from an infection by said virus of interest is enhanced by a replicon, virus and/or nucleic acid sequence according to the invention, resulting in a better counteraction of disease.
Replicons, viruses and nucleic acid sequences according to the invention are thus suitable for use as a medicament and/or vaccine. A replicon or virus according to the present invention for use as a medicament and/or vaccine is therefore herewith provided, as well as an isolated or recombinant nucleic acid sequence according to the invention for use as a medicament and/or vaccine. It is possible to place at least one HIV sequence essential for replication under direct or indirect control of an rtTA and/or sc rtTA nucleic acid of the present invention. This way, controlled replication of HIV has become possible allowing for at least partial prophylaxis and/or treatment of AIDS. A use of an isolated or recombinant replicon, virus and/or nucleic acid sequence according to the invention for the preparation of a medicament or immunogenic composition for at least in part preventing and/or treating AIDS is therefore also herewith provided.
One further embodiment provides an isolated cell comprising a nucleic acid sequence, a replicon and/or a virus according to the invention.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
We have previously reported on the construction of an infectious HIV-rtTA virus that is critically dependent on dox for replication (Verhoef et al. 2001; Das et al. 2004b; Marzio et al. 2001). In this virus, the natural transcriptional activator Tat and its TAR binding site were inactivated by mutation and functionally replaced by the components of the Tet-on system (
We started multiple, independent virus cultures of the HIV-rtTAF86Y A209T variant, which contains both the optimized LTR-tetO promoter configuration and the improved rtTA gene. After culturing the virus with dox for up to 200 days, the rtTA gene was sequenced. The F86Y and A209T mutations were stably maintained in all analyzed cultures. Several viruses from independent cultures had acquired additional mutations in the rtTA gene. A virus variant should have a replication advantage to become the dominant sequence in a virus population. Mutations in rtTA may improve rtTA function, and thus enhance viral replication. To increase the chance of identifying such beneficial mutations, we focused on the rtTA mutations that were observed in multiple cultures (
Characterization of the Evolved rtTA Variants.
To test whether the evolved rtTAs exhibit improved transcriptional activity, we assayed rtTA activity in a regular Tet system. Expression plasmids encoding the original (wild-type, this is the rtTA2S-S2 variant described in Urlinger et al. 2000) and mutant rtTA proteins (V1-V10, Table 1) were constructed and transfected into C33A cells with a plasmid expressing luciferase reporter under the control of the viral LTR-2ΔtetO promoter. The luciferase level measured two days after transfection reflects rtTA activity (
To test whether this rtTA optimization reflects a specific adaptation to the viral LTR-2ΔtetO promoter, we assayed rtTA activity with a reporter gene construct in which luciferase expression is under the control of a minimal CMV promoter coupled to an array of seven tetO elements [4]. All evolved rtTA variants demonstrate improved activity with this promoter construct (
To compare the dox-sensitivity of the rtTA variants in another way, we calculated the dox concentration that each rtTA variant needs to reach an activity similar to that of the wild-type rtTA at 1000 ng/ml dox (
Combining the Beneficial Mutations Further Improves rtTA Activity.
Analysis of the evolved rtTA variants revealed that the double mutants exhibit a higher activity and dox-sensitivity than the single mutants. For instance, V6 (R171K) is 4.4-fold more sensitive than the wild-type rtTA, and the double mutant V9 (V9I R171K) is 14.9-fold more dox-sensitive. We therefore constructed additional rtTA variants in which the observed mutations were combined (V11-V18, Table 1), and assayed their activity (
A more extensive list of novel rtTA variants that carry mutations observed in HIV-rtTA evolution and that demonstrate improved transcriptional activity and dox-sensitivity is shown in
To exclude the possibility that the enhanced activity observed for the mutant rtTAs resulted from an increased protein level, we determined the intracellular steady state level of the rtTA proteins. Lysates of HeLa X1/6 cells transfected with rtTA expression plasmids were subjected to SDS-PAGE followed by Western blot analysis with polyclonal anti-TetR antibodies. An equal amount of rtTA protein was detected for all naturally evolved and constructed variants (
Novel rtTA Variants can be Activated by Dox-Like Compounds.
Dox is the most efficient effector that controls the Tet-on system. Other dox-like compounds, such as tetracycline (Tc) and minocycline (Mc), do not effectively activate the wild-type rtTA and the original HIV-rtTA virus. To test if the novel rtTA variants with improved activity and dox-sensitivity have a broader effector-specificity, we assayed the activity of a subset of these rtTA variants at different Tc and Mc concentrations (
rtTA Variants Improve HIV-rtTA Replication.
To test if the rtTA variants with enhanced activity and dox-sensitivity can also improve HIV-rtTA replication, we constructed viral variants with the rtTA genes encoding either mutant V7 or V14, and assayed their replication in SupT1 cells at different dox concentrations (
We also assayed replication of these new HIV-rtTA variants in the presence of Tc and Mc (
Amino acid substitutions in rtTA at position 9, 19, 37, 67, 68, 86, 138, 157, 171, 177, 195 and/or 209, which are observed during evolution of the HIV-rtTA virus, enhance the transcriptional activity and/or inducer-sensitivity of rtTA. Moreover, these mutations broaden the inducer-specificity of rtTA. The most optimal rtTA variants (V15 and V16) are 7-fold more active at high dox levels and 100-fold more sensitive to dox than the original rtTA. Importantly, these rtTA variants do not show any basal activity in the absence of dox. High activity and dox-sensitivity of these novel rtTAs significantly improve the performance of the Tet-on system.
Cell cultures. The human T-lymphocyte cell line SupT1 (Smith et al. 1984) was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 U/ml). HeLa X1/6 (Baron et al. 1997) is a HeLa-derived cervix carcinoma cell line, containing chromosomally integrated copies of the CMV-7tetO promoter/luciferase reporter construct pUHC13-3 (Gossen et al. 1992). HeLa X1/6 and C33A cervix carcinoma cells (ATCC HTB31) (Auersperg, 1964) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, minimal essential medium nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 U/ml). All cell cultures were kept at 37° C. and 5% CO2.
Virus replication. Construction of the HIV-rtTA molecular clone was described previously (Verhoef et al. 2001; Das et al. 2004b). The HIV-rtTA variant used in this study contains the 2ΔtetO configuration (Marzio et al. 2001; Marzio et al. 2002) in both the 5′ and the 3′ LTR. SupT1 cells (5×106) were transfected with 5 μg of the HIV-rtTA molecular clone by electroporation (250 V and 975 μF). Viral replication was induced with doxycycline (dox, D-9891, Sigma, St. Louis, Mo., USA), tetracycline (Tc, Sigma T-3383) or minocycline (Mc, Sigma M-9511). The CA-p24 level in the cell-free culture supernatant was determined by antigen capture enzyme-linked immunosorbent assay (ELISA) (Back et al. 1996).
For the selection of evolved viruses, SupT1 cells were transfected with the HIV-rtTA-F86Y A209T molecular clone (Das et al. 2004a), and cultured in the presence of 1 μg/ml dox for up to 200 days. The virus containing culture supernatant was passaged onto fresh SupT1 cells at the peak of infection, as determined by the massive appearance of syncytia. At regular intervals, cell and supernatant samples were taken from the culture and stored at −80° C. for subsequent analysis.
Proviral DNA analysis and cloning of evolved sequences. Total cellular DNA from infected cells was isolated as described previously (Das et al. 1997). The proviral rtTA genes were PCR amplified with the sense primer tTA1 (5′-ACAGCCATAGCAGTAGCTGAG-3′) and the antisense primer tTA-rev2 (5′-GATCAAGGATATCTTGTCTTCGT-3′), and sequenced with the bigdye terminator cycle sequencing kit (Applied Biosystems, Foster city, CA, USA). The PCR products were digested with XbaI and SmaI and used to replace the corresponding fragment in pCMV-rtTA, in which the expression of wild-type rtTA (rtTA2S-S2, (Urlinger et al. 2000)) is controlled by the human cytomegalovirus (CMV) immediate-early promoter. Mutant rtTA genes were cloned from pCMV-rtTA into the shuttle vector pBlue3′LTRext-deltaU3-rtTAF86Y A209T-2ΔtetO (Das et al. 2004a) using the XcmI and NdeI sites and subsequently cloned back into the HIV-rtTA molecular clone as BainHI-BglI fragments. To introduce the F67S and G138D mutations into evolved rtTA variants, mutagenesis PCR (Mikaelian et al. 1992) was performed with the corresponding pCMV-rtTA plasmid and the mutagenic primer (primer M) tTA-F67S (5′-CATACCCACTCCTGCCCCCTGGAAGGCGA-3′, mismatching nucleotide underlined) or tTA-G138D (5′-GTCCGCCGTGGACCACTTTACACTGGGCT-3′) and the general primers 5′-TGGAGACGCCATCCACGCT-3′ (primer 1), 5′-TGAAATCGAGTTTCTCCAGGCCACATATGA-3′ (primer 2), and 5′-TCACTGCATTCTAGTTGTGGT-3′ (primer 3). Briefly, PCR reactions were performed with primer M plus primer 3, and with primer 1 plus primer 2. The PCR products were purified, mixed, and PCR amplified with primers 1 and 3 (see reference (Mikaelian et al. 1992) for details). The resulting mutated rtTA genes were cloned as EcoRI-BamHI fragments into pCMV-rtTA. All constructs were verified by sequence analysis.
rtTA activity assay. Two firefly luciferase reporter constructs with different promoter configurations were used. pLTR-2ΔtetO-luc contains the LTR-2ΔtetO promoter derived from the HIV-rtTA molecular clone (Marzio et al. 2001; Marzio et al. 2002). pCMV-7tetO-luc, previously named pUHC13-3 (Gossen et al. 1992), contains seven tetO elements located upstream of a minimal CMV promoter. The plasmid pRL-CMV (Promega, Madison, Wis., USA), in which the expression of Renilla luciferase is controlled by the CMV promoter, was used as an internal control to allow correction for differences in transfection efficiency.
C33A and HeLa X1/6 cells were grown in 2-cm2 wells to 60% confluency and transfected by the calcium phosphate precipitation method (Das et al. 1999). C33A cells were transfected with 0.4 ng pCMV-rtTA (wild-type or mutant), 20 ng pLTR-2ΔtetO-luc or pCMV-7tetO-luc, 0.5 ng pRL-CMV, and 980 ng pBluescript as carrier DNA. HeLa X1/6 cells were transfected with 8 ng pCMV-rtTA, 2.5 ng pRL-CMV, and 990 ng pBluescript. The amount of the DNA constructs was optimized for each cell type to keep rtTA-mediated transactivation within the linear range and to avoid squelching of transcription factors. Cells were cultured for 48 hours in the presence of different concentrations of dox, Tc or Mc, and lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined with the dual-luciferase reporter assay (Promega). The activity of the rtTA variants was calculated as the ratio of the firefly and Renilla luciferase activities, and corrected for between-session variation.
Western blot analysis. HeLa X1/6 cells were transfected at 90% confluency with 1 μg of wild-type or mutant pCMV-rtTA and 2 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA) in 2-cm2 wells. Cells were cultured for 48 hours and lysed in 100 μl of Passive Lysis Buffer. 10 μl of the lysate was subjected to SDS-polyacrylamide gel separation, and transferred to Immobilon-P membrane (Millipore, Billerica, Mass., USA). For immunochemical detection of rtTA variants, membranes were incubated with rabbit serum containing polyclonal anti-TetR antibodies (Krueger et al. 2003). Bound antibodies were visualized with peroxidase-linked anti-rabbit IgG and the ECL+ kit (Amersham Biosciences, Freiburg, Germany) and analyzed with a Storm 860 Imager (Amersham Biosciences).
HIV-1 vaccines based on a live-attenuated virus have shown promise in the SIV-macaque model, but are generally considered unsafe for use in humans. The major safety concern is that chronic low-level replication of the attenuated virus may eventually lead to selection of fitter and more pathogenic virus variants. Ideally, one would like to restrict replication of a vaccine virus to the time window that is needed to elicit a protective immune response. We previously presented a novel vaccine approach that uses a conditional-live HIV-1 virus. In this HIV-rtTA virus, the viral transcriptional activator Tat and its TAR binding site were inactivated by mutation and functionally replaced by the components of the Tet-on system. This system, in which gene expression is stringently controlled by the non-toxic effector doxycycline (dox), is widely applied to regulate gene expression in eukaryotes. The rtTA gene encoding the transcriptional activator was inserted in place of the nef gene, and the tet-operator (tetO) DNA binding sites were placed in the viral LTR promoter. This HIV-rtTA virus does not replicate in the absence of dox. Binding of dox to rtTA triggers a conformational change that allows the protein to bind tetO DNA, resulting in transcriptional activation and subsequent viral replication. Upon vaccination with this virus, replication can be temporarily activated and controlled to the extent needed for induction of the immune system by transient dox administration.
The potential use of this dox-dependent HIV-rtTA virus as a vaccine raises new safety questions concerning the genetic stability of the introduced Tet-on system. There are several hypothetical evolutionary routes toward a constitutively replicating virus. First, the virus may restore the function of the Tat-TAR system, despite the multiple inactivating mutations that were introduced in both elements to avoid simple reversion to the wild-type sequence. In this scenario, the dox-controlled rtTA-tetO system will become superfluous, and may be inactivated over time by mutation or deletion. Second, the viral LTR promoter could become a constitutive transcription element, for instance by acquisition of a binding site for a constitutively expressed cellular transcription factor. Replication of such a virus is not dependent on a virally encoded transactivator, neither Tat nor rtTA. Third, the introduced rtTA-tetO axis may lose dox-dependence, thereby creating an uncontrolled Tet system. This scenario would for instance occur through acquired mutations in the rtTA protein that shift its conformation into the DNA-binding mode, even in the absence of dox.
To address these safety issues, we followed the evolution of HIV-rtTA in multiple, independent virus cultures. We observed loss of dox-control in several cultures, which in all cases resulted from a typical amino acid substitution either at position 19 or 37 in the rtTA protein. We developed novel rtTA variants with alternative amino acids at these positions, and demonstrated that the corresponding HIV-rtTA variants did not lose dox-control in long-term cultures. Thus, we improved the genetic stability of the Tet-on system and the HIV-rtTA vaccine candidate by blocking unwanted evolutionary routes.
Virus cultures. The HIV-rtTA infectious molecular clone is a derivative of the HIV-1 LAI proviral plasmid (Peden et al. 1991) and was described previously (Das et al. 2004b; Verhoef et al. 2001). HIV-rtTA used in this study is the KYK version, which contains the inactivating Y26A mutation in the Tat gene and five nucleotide substitutions in the TAR hairpin motif. This virus contains the rtTA2S-S2 gene (Urlinger et al. 2000) in place of the nef gene and eight tetO sequences in the LTR promoter region. The HIV-rtTA 2ΔtetO clone is identical to HIV-rtTA, but with the optimized 2ΔtetO promoter configuration (Marzio et al. 2001; Marzio et al. 2002). HIV-rtTAF86Y A209T contains the LTR-2ΔtetO promoter and the recently described rtTAF86Y A209T gene (Das et al. 2004a).
SupT1 T cells were grown at 37° C. and 5% CO2 in RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. SupT1 cells were transfected with HIV-rtTA molecular clones by electroporation. Briefly, 5×106 cells were washed in RPMI1640 with 20% FBS and mixed with 5 μg of DNA in 250 μl RPMI1640 with 20% FBS. Cells were electroporated in 0.4-cm cuvettes at 250 V and 975 μF and subsequently resuspended in RPMI1640 with 10% FBS. The CA-p24 level in the cell-free culture supernatant was determined by antigen capture enzyme-linked immunosorbent assay (ELISA) (Back et al. 1996).
The 24-well evolution experiment was started with transfection of 40 μg of the HIV-rtTA proviral plasmid into 2×107 SupT1 cells. Cells were split into 24 independent cultures and maintained in the presence of 1 μg/ml dox (Sigma D-9891) for up to 200 days. The virus containing culture supernatant was passaged onto fresh SupT1 cells at the peak of infection, as determined by the massive appearance of syncytia. At regular intervals, supernatant samples were taken from the culture and tested in parallel infections with and without dox. Cell samples were stored at −80° C. for subsequent analysis.
HIV-rtTA infected cells were pelleted by centrifugation and washed with phosphate-buffered saline. DNA was solubilized by resuspending the cells in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA-0.5% Tween 20, followed by incubation with 200 μg/ml of proteinase K at 56° C. for 60 min and 95° C. for 10 min. Proviral DNA sequences were PCR amplified from total cellular DNA. The first exon of the Tat gene was amplified with the primers KV1 (5′-CCATCGATACCGTCGACATAGCAGAATAGG-3′) and 3'TAT (5′-CGGGAATTCTTACTGCTTTGATAGAGAAAC-3′). The LTR-tetO region was amplified with the primers tTA-tetO1 (5′-CTCCCCGGGTAACTAAGTAAGGAT-3′) and C(N1) (5′-GGTCTGAGGGATCTCTAGTTACCAGAGTC-3′). The rtTA gene was amplified with the primers tTA1 (5′-ACAGCCATAGCAGTAGCTGAG-3′) and tTA-rev2 (5′-GATCAAGGATATCTTGTCTTCGT-3′). All PCR fragments were sequenced with the bigdye terminator cycle sequencing kit (Applied Biosystems). For the cloning of the G19E or E37K mutated rtTA sequences into the HIV-rtTA provirus, rtTA PCR fragments were digested with XcmI and SmaI and cloned into the corresponding sites of the shuttle vector pBlue3′LTRext-deltaU3-rtTA-2ΔtetO (16). The BamHI-BglI fragment of the shuttle vector was used to replace the corresponding sequence in HIV-rtTA 2ΔtetO.
Construction of Novel HIV-rtTA Variants and rtTA Expression Plasmids.
HIV-rtTA variants with an alternative G codon (GGU instead of GGA) at rtTA position 19 and with a wild-type (E) or alternative amino acid (D, F, L, N, Q, R, S) at position 37 were constructed by oligonucleotide directed mutagenesis.
The oligonucleotide G19 (5′-ATAACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTG GAATTACTCAATGGTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGC T-3′, mutated nucleotide underlined) was annealed to the oligonucleotide rev-37 (5′-AGCAGGGCCCGCTTGTTCTTCACGTGCCAGTACAGGGTAGGCTGXXXA ACTCCCAGCTTTTGAGCGAGTTTCCTTGTCGTCAGGCCTTCGA-3′, with XXX corresponding to amino acid 37; this triplet is CTC for E, ATC for D, GAA for F, AAG for L, ATT for N, CTG for Q, GCG for R, and AGA for S), both strands were completed with Klenow DNA polymerase in the presence of dNTPs, digested with XcmI and ApaI, and ligated into the similarly digested shuttle vector pBlue3′LTRext-deltaU3-rtTAF86Y A209T-2ΔtetO (Das et al. 2004a). The BamHI-BglI fragment of the shuttle vector was used to replace the corresponding sequence in HIV-rtTA 2ΔtetO.
The plasmid pCMV-rtTA contains the rtTA2S-S2 gene in the expression vector pUHD141-1/X (Urlinger et al. 2000). To generate rtTA variants with different amino acids at position 19 or 37, PCR was performed on pCMV-rtTA with the sense primer random-rtTA-19 (5′-TTCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTG GAATTACTCAATNNKGTCGGTATCGAAGGCCTGACGA-3′, mutated nucleotide underlined with K corresponding to T or G, and N corresponding to T, C, A or G) plus the antisense primer CMV2 (5′-TCACTGCATTCTAGTTGTGGT-3′) or with the sense primer CMV1 (5′-TGGAGACGCCATCCACGCT-3′) plus the antisense primer random-rtTA-37 (5′-AGCAGGGCCCGCTTGTTCTTCACGTGCCAGTACAGGGTAGGCTGMNN AACTCCCAGCTTTTGAGCGA-3′, mutated nucleotide underlined with M corresponding to A or C, and N corresponding to T, C, A or G), respectively. The mutated rtTA sequences were cloned as XbaI-ApaI fragments into pCMV-rtTAF86Y A209T (Das et al. 2004a). All constructs were verified by sequence analysis. To combine the G19F (UUU codon) and E37L (CUU codon) mutations, the E37L-containing StuI-BamHI fragment of pCMV-rtTAE37L was used to replace the corresponding sequence in pCMV-rtTAG19F, resulting in pCMV-rtTAG19F E37L. The rtTAG19F E37L sequence was cloned into the shuttle vector pBlue3′LTRext-deltaU3-rtTAF86Y A209T-2ΔtetO (Das et al. 2004a) using the XcmI and NdeI sites and subsequently cloned into the HIV-rtTA 2ΔtetO molecular clone as a BamHI-BglI fragment.
rtTA Activity Assay.
HeLa X1/6 cells (Baron et al. 1997) are derived from the HeLa cervix carcinoma cell line and harbor chromosomally integrated copies of the CMV-7tetO firefly luciferase reporter construct pUHC13-3 (Gossen et al. 1992). Cells were grown at 37° C. and 5% CO2 as a monolayer in Dulbecco's modified Eagle's medium supplemented with 10% FBS, minimal essential medium nonessential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin.
HeLa X1/6 cells were grown in 2-cm2 wells to 60% confluency and transfected by the calcium phosphate precipitation method. 1 μg of DNA mixture in 15 μl water was mixed with 25 μl of 50 mM HEPES (pH 7.1)-250 mM NaCl-1.5 mM Na2HPO4 and 10 μl of 0.6 M CaCl2, incubated at room temperature for 20 min and added to the culture medium. The DNA mixture consisted of 8 ng pCMV-rtTA, 2.5 ng pRL-CMV, and 990 ng pBluescript as carrier DNA. The plasmid pRL-CMV (Promega), in which the expression of Renilla luciferase is controlled by the CMV promoter, was used as an internal control to allow correction for differences in transfection efficiency. Cells were cultured after transfection for 48 hours at different dox concentrations and then lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined with the Dual-Luciferase Reporter Assay (Promega). The expression of firefly and Renilla luciferase was within the linear range and no squelching effects were observed. The activity of the rtTA variants was calculated as the ratio of the firefly and Renilla luciferase activities, and corrected for between-session variation (Retrovirology, submitted).
Appearance of HIV-rtTA variants with reduced dox-dependence. We have previously reported on the construction of a conditional-live HIV-1 variant (Das et al. 2004b; Verhoef et al. 2001), in which the natural Tat-TAR elements that control viral gene expression and replication were inactivated by mutation and functionally replaced by the rtTA-tetO elements of the Tet-on system for inducible gene expression (
The replication curves of the original HIV-rtTA virus and two representative dox-independent virus cultures are shown in
Amino Acid Substitutions in rtTA Confer the Loss of Dox-Control.
To demonstrate that these rtTA mutations are responsible for the observed viral replication without dox, we constructed HIV-rtTA molecular clones with the G19E or E37K mutation in the rtTA gene and assayed their replication at different dox concentrations (
The results described above were obtained with the original HIV-rtTA virus, which replicates relatively poor. We also tested the genetic stability of two improved HIV-rtTA variants in a similar 24-well long-term culture assay. HIV-rtTA 2ΔtetO is identical to HIV-rtTA, but with the improved LTR-2ΔtetO promoter (Marzio et al. 2001; Marzio et al. 2002), and HIV-rtTAF86Y A209T contains in addition the improved rtTAF86Y A209T gene (Das et al. 2004a). With both viruses we again observed the appearance of variants that replicated without dox, albeit at a significantly slower rate compared with the original HIV-rtTA (
HIV-rtTA Variants with Alternative Codons at rtTA Positions 19 and 37.
In the evolution experiments, we observed very specific amino acid substitutions that reduced dox-dependence at only two rtTA positions (G19E and E37K). The introduction of alternative rtTA codons may make such specific amino acid substitutions more difficult or even prevent these unwanted evolutionary routes. For instance, the G19E mutation involves a GGA to GAA codon change, and the G-to-A transition is the most frequent error during HIV-1 reverse transcription. Introduction of an alternative Gly codon (GGU or GGC) would require a much more difficult two-hit mutation, including one transversion, to create a Glu codon (GAA or GAG). Such a difference in the mutational frequency strongly influences the course of HIV-1 evolution.
A similar strategy is not possible for E37K because all possible Glu codons (GAA and GAG) require only a single G-to-A mutation to turn into a Lys codon (AAA or AAG). As an alternative blocking strategy, we could replace the E37-codon with a non-Glu codon that would be more difficult to transform into a Lys codon. However, such an amino acid substitution should ideally not affect the activity or dox-dependence of the rtTA protein. We first examined natural variation at this position in the Tet repressor (TetR). The rtTA protein is based on the E. coli class B TetR (TetRB), but there are six additional TetR classes (A, C-E, G, H). TetR from class D, E and H also have the Glu at position 37, but TetR from class A, C and G have a Gln instead. Evolution of a Gln codon (CAA or CAG) to a Lys codon (AAA or AAG) would require only a single C-to-A mutation, but this transversion is less frequently observed in HIV-1 evolution. We therefore constructed an HIV-rtTA variant with a Gln codon (CAG) at position 37 (E37Q). In addition, we constructed variants with either an Asp (GAU; E37D), Asn (AAU; E37N), Ser (UCU; E37S), Arg (CGC; E37R), Phe (UUC; E37F) or a Leu codon (CUU; E37L). The E37D substitution leaves the acidic nature of the residue intact. The E37N and E37S mutations, like the natural variant E37Q, result in polar, uncharged residues. The E37F and E37L mutations result in hydrophobic residues. The E37R substitution creates a basic residue that is similar to the E37K mutation selected through viral evolution. When allowed by the degeneracy of the genetic codon, we chose the codon that requires most mutations to convert into a Lys codon. For example, a CGC rather than an AGA codon was chosen for the E37R variant. Moreover, all new HIV-rtTA variants contain the alternative Gly codon (GGU) at position 19.
We tested replication of these novel HIV-rtTA variants in SupT1 cells with and without dox (
Testing all Possible Position 37 Variants of rtTA.
We constructed rtTA expression plasmids with all possible amino acids at position 37. The activity of these variants was assayed by transfection into HeLa X1/6 cells (Baron et al. 1997) that contain stably integrated copies of the CMV-7tetO luciferase reporter construct (Gossen et al. 1992). Transfected cells were cultured for two days in the presence of 0-1000 ng/ml dox. We subsequently determined the intracellular luciferase level, which reflects rtTA activity. As shown in
Comparison of the rtTA activity data (
In the codon table, every change in row or column represents a single nucleotide substitution. This colored codon table (
Testing all Possible Position 19 Variants of rtTA.
Like the E37K mutation, the G19E mutation causes viral replication in the absence of dox. To reveal whether other amino acid substitutions at this position would similarly result in a loss of dox-dependence, we constructed rtTA expression plasmids with all possible amino acids at position 19. The activity of these rtTA variants was analyzed as described above for the position 37 variants. As shown in
rtTA with Safety-Lock Mutations Prevents the Loss of Dox-Control.
We constructed an rtTA variant that combines the two safety-lock mutations: Phe (UUU) at position 19 (G19F) and Leu (CUU) at position 37 (E37L). This rtTA variant shows very low basal activity (less than 0.1%) and its activity gradually increases with increasing dox levels (
rtTA Variants with an Alternative Amino Acid at Position 19 or 37 Demonstrate an Improved Transcriptional Activity and Dox-Sensitivity.
Most rtTA variants with an alternative amino acid at position 19 (alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine or tyrosine) and some of the rtTA variants with an alternative amino acid at position 37 (cysteine, methionine, glutamine, arginine or threonine) show an increased transcriptional activity at a low dox concentration and/or an increased transcriptional activity at a high dox concentration when compared with the original (wild-type) rtTA (
When currently known rtTA is incorporated in a replicating system (e.g. in a replicon), rtTA is at risk of losing dox-control due to mutations at rtTA amino acid position 19 and/or 37 acquired during evolution of the system. Such undesired evolution is prevented by the introduction of alternative codons at these amino acid positions. Preferred alternative codons require multiple nucleotide substitutions to convert into a codon encoding an amino acid that would mediate loss of dox-control of rtTA. As an example we demonstrate that a phenylalanine codon (UUU) at rtTA amino acid position 19 and a leucine codon (CUU) at position 37 improve the genetic stability of rtTA and prevent at least in part the loss of dox-control. Our results demonstrate that other amino acid codons at position 19 (encoding alanine, cysteine, phenylalanine, histidine, isoleucine, leucine, methionine, asparagine, arginine, serine, threonine, valine, tryptophane or tyrosine) and position 37 (encoding histidine, leucine or arginine) similarly improve the genetic stability of rtTA.
The introduction of alternative amino acids at rtTA amino acid position 19 and/or 37 improve the transcriptional activity and/or inducer-sensitivity of rtTA. Specifically, introduction of an alanine, cysteine, aspartate, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine or tyrosine at rtTA amino acid position 19, and/or the introduction of a cysteine, methionine, glutamine, arginine or threonine at rtTA amino acid position 37 results in an increased transcriptional activity and/or dox-sensitivity of rtTA.
Single-chain Tet transregulators have recently been developed, in which two TetR domains are connected by a peptide linker and one VP16 activation domain or KRAB repressor domain is positioned at the C-terminal end (Krueger et al. 2003). These transregulators fold intramolecularly and do not dimerize with each other. Unfortunately, the single-chain version of rtTA (sc rtTA) exhibits reduced activity when compared with the regular rtTA, and this low activity may thwart its use in applications that require an active Tet-on system.
We have incorporated the rtTA gene and the tetO elements into the HIV-1 genome to control virus replication. During culturing of this dox-dependent virus, spontaneous viral evolution selected for improved virus variants, in which the introduced Tet-on system was optimized. We have identified several amino acid substitutions in the rtTA gene that greatly enhance the transcriptional activity and dox-sensitivity of the transactivator. To test whether these mutations similarly improve other TetR-based transactivators, we introduced them into sc rtTA. All mutations enhanced sc rtTA activity. The most active sc rtTA variant is ˜30-fold more active than the original sc rtTA, and is almost as active as the regular rtTA.
Construction of sc rtTA variants. The plasmids pCMV-rtTA and pCMV-scrtTA contain the rtTA2S-S2 and sc rtTA2-S2 genes, respectively, cloned in the expression vector pUHD141-1/X (Krueger et al. 2003; Urlinger et al. 2000). The sc rtTA gene contains two TetR domains and a single activation domain. To introduce mutations into the N-terminal TetR domain, the EcoRI-BfuAI fragment of pCMV-scrtTA was replaced with the corresponding fragment of the appropriate pCMV-rtTA plasmid. Mutations were introduced into the C-terminal TetR domain of sc rtTA by mutagenesis PCR (Mikaelian et al. 1992) on pCMV-scrtTA with the mutagenic primers (primer M) scrtTA-V9I (5′-GGCTCTAGATCTCGTTTAGATAAAAGTAAAATCATTAACAGCGCA-3′), scrtTA-F67S (5′-AGGCACCATACTCACTCTTGCCCTTTA-3′), scrtTA-F86Y (5′-AACGCTAAAAGTTATAGATGTGCT-3′), or scrtTA-G138D (5′-CAGCGCTGTGGACCACTTTACTTTA-3′) and the primers 5′-TAATCATATGTGGCCTGGAGAA-3′ (primer 1), 5′-AGGCGTATTGATCAATTCAAGGCCGAATAAG-3′ (primer 2), and 5′-TCACTGCATTCTAGTTGTGGT-3′ (primer 3) as described above for the tTA mutations. The final PCR products were digested with BglII and SmaI and used to replace the corresponding fragment of pCMV-scrtTA. All constructs were verified by sequence analysis.
Cell culture and rtTA activity assay. The activity of rtTA and sc rtTA was assayed in HeLa X1/6 cells (Baron et al. 1997), which are HeLa-derived cells containing chromosomally integrated copies of the CMV-7tetO luciferase reporter construct pUHC13-3 (Gossen et al. 1992). Cells were cultured at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, minimal essential medium nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were grown in 2-cm2 wells to 60% confluency and transfected with the pCMV-rtTA or pCMV-scrtTA expression plasmids and the plasmid pRL-CMV (Promega) by the calcium phosphate precipitation method. pRL-CMV expresses Renilla luciferase from the CMV promoter and was used as an internal control to allow correction for differences in transfection efficiency. 1 μg of DNA mixture in 15 μl water was mixed with 25 μl of 50 mM HEPES (pH 7.1)-250 mM NaCl-1.5 mM Na2HPO4 and 10 μl of 0.6 M CaCl2, incubated at room temperature for 20 min, and added to the culture medium. The DNA mixture consisted of 20 ng pCMV-scrtTA or pCMV-rtTA, 2 ng pRL-CMV, and 978 ng pBluescript for sc rtTA or rtTA activity assay. Cells were cultured after transfection for 48 hours at different dox (D-9891, Sigma) concentrations and then lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined with the Dual-Luciferase Reporter Assay (Promega). The expression of firefly and Renilla luciferase was within the linear range and no squelching effects were observed. The activity of the transactivators was calculated as the ratio of the firefly and Renilla luciferase activities, and corrected for between-session variation.
Mutations Observed in rtTA Improve Sc rtTA Activity.
In sc rtTA, two TetR domains are connected head to tail by a peptide linker, and a single activation domain is fused to the C-terminal TetR domain. The mutations that did improve rtTA activity are all positioned within the TetR domain of the protein (
Similar results were obtained upon introduction of the mutations into the C-terminal TetR domain of sc rtTA (
Introduction of the mutations in both TetR domains resulted in the most active sc rtTA variants (
We have identified amino acid substitutions in rtTA that greatly improve the transcriptional activity and dox-sensitivity of the transactivator. In this Example, we tested whether these mutations similarly affect sc rtTA. Our results demonstrate that all mutations did significantly enhance sc rtTA activity. Both the transactivators rtTA and sc rtTA are activated by doxycycline. Our results demonstrate that the sc rtTA activity is significantly improved by introduction of at least one mutation that enhances rtTA activity. The most active sc rtTA variant in this study was obtained by introducing beneficial mutations in both TetR domains. However, sc rtTA is also improved by at least one mutation in only one of the TetR domains. The sc rtTA variants with beneficial mutations in the N-terminal TetR domain appear to be more active than the variants with the same mutations in the C-terminal TetR domain.
The sc rtTA variant with the F67S and F86Y mutations in both TetR domains is ˜30-fold more active than the original sc rtTA at high dox levels, and does not show any background activity in the absence of dox. This novel sc rtTA is almost as active and dox-sensitive as rtTA, and is therefore suitable for replacing the regular rtTA in applications where multiple TetR-based regulatory systems are used in the same cell or organism.
The transcriptional activity and inducer-sensitivity of single chain rtTA activity is significantly improved by the introduction of amino acid substitutions that were found by us to improve the transcriptional activity and inducer-sensitivity of rtTA. We thus for instance generated sc rtTA variants with an up to ˜30-fold increased transcriptional activity and an increased dox-sensitivity by the introduction of a F86Y, a V9I, a F67S and/or a G138D amino acid substitution into the original sc rtTA.
We have demonstrated that long-term replication of HIV-rtTA resulted in virus variants that no longer depend on dox for replication. This reduced dox-dependence was associated with an amino acid substitution in the rtTA protein either at position 19 (glycine to glutamate; G19E) or at position 37 (glutamate to lysine; E37K). We developed an HIV-rtTA variant with safety-lock mutations (G19F and E37L) in the rtTA gene to block these undesired evolutionary routes. This novel variant showed improved genetic stability and did not lose dox-control in long-term cultures with dox (see Example 2).
As a vaccine, replication of HIV-rtTA would be temporally switched on to induce anti-viral immune responses. Subsequent dox-withdrawal impose alternative evolutionary pressure on the virus than long-term culturing with dox. Specifically, there is a risk of rtTA variants with a tTA-like phenotype, which are active without dox and inhibited by dox, appearing in dox-washout experiments, whereas such variants are counter selected in the presence of dox. We therefore followed evolution of HIV-rtTA in multiple, independent virus cultures that were transiently activated by dox. The virus did indeed lose dox-control in a significant number of cultures after dox-withdrawal. We identified a typical amino acid substitution at position 56 in the rtTA protein, which was found to be responsible for the dox-independent replication. This mutation had never been observed in long-term cultures with dox. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus improves the genetic stability and safety of HIV-rtTA.
Virus cultures. The HIV-rtTA infectious molecular clone is a derivative of the HIV-1 LAI proviral plasmid (Peden et al, 1991) and was described previously (Das et al, 2004b; Verhoef et al, 2001). HIV-rtTA used in this study contains the inactivating Y26A mutation in the Tat gene, five nucleotide substitutions in the TAR hairpin motif, the rtTAF86Y A209T gene (Das et al, 2004a) in place of the nef gene, and the LTR-2ΔtetO promoter configuration (Marzio et al, 2001; Marzio et al, 2002).
SupT1 T cells were cultured at 37° C. and 5% CO2 in RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. SupT1 cells were transfected with HIV-rtTA molecular clones by electroporation. Briefly, 5×106 cells were washed in RPMI1640 with 20% FBS and mixed with 5 μg of DNA in 250 μl RPMI1640 with 20% FBS. Cells were electroporated in 0.4-cm cuvettes at 250 V and 975 μF and subsequently resuspended in RPMI1640 with 10% FBS. The CA-p24 level in the cell-free culture supernatant was determined by antigen capture enzyme-linked immunosorbent assay (ELISA) (Back et al, 1996).
The evolution experiment was started with transfection of 15 μg HIV-rtTA proviral plasmid into 1.5×107 SupT1 cells. Cells were split into 12 independent cultures and dox (Sigma D-9891) was added to initiate viral replication. Three days after transfection, dox was removed from the cultures by washing the cells twice with medium, each followed by a 30 min incubation at 37° C. and 5% CO2 to allow release of dox from cells. Cells were subsequently resuspended in medium and cultured without dox. If virus replication was apparent as indicated by the formation of syncytia, the virus containing culture supernatant was passaged onto fresh SupT1 cells. Infected cell samples were used to analyze the proviral rtTA sequence.
Proviral DNA analysis of evolved sequences. HIV-rtTA infected cells were pelleted by centrifugation and washed with phosphate-buffered saline. Total cellular DNA was solubilized by resuspending the cells in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA-0.5% Tween 20, followed by incubation with 200 μg/ml of proteinase K at 56° C. for 60 min and 95° C. for 10 min. The proviral rtTA genes were PCR amplified with primers tTA1 (5′-ACAGCCATAGCAGTAGCTGAG-3′) and tTA-rev2 (5′-GATCAAGGATATCTTGTCTTCGT-3′), and sequenced with the bigdye terminator cycle sequencing kit (Applied Biosystems).
Construction of novel rtTA expression plasmids and HIV-rtTA variants. The plasmid pCMV-rtTA contains the rtTA2S-S2 gene in the expression vector pUHD141-1/X (Urlinger et al, 2000). To introduce the P56S mutation, the proviral PCR product with this mutation was digested with XbaI and SmaI and used to replace the corresponding fragment in pCMV-rtTA. To generate rtTA variants with the G19F and E37L mutations and different amino acids at position 56, mutagenesis PCR was performed on pCMV-rtTAG19F E37L (example 2) with the sense primer random-rtTA-56 (5′-AAGCGGGCCCTGCTCGATGCCCTGNNKATCGAGATGCTGGACAGGC-3′, with K corresponding to G or T, and N corresponding to G, A, T or C) plus the antisense primer CMV2 (5′-TCACTGCATTCTAGTTGTGGT-3′). Mutant rtTA sequences were cloned as ApaI-BamHI fragments into pCMV-rtTAG19F E37L. Novel rtTA sequences were cloned into the shuttle vector pBlue3′LTRext-deltaU3-rtTAF86Y A209T-2ΔtetO (Das et al, 2004a) using the XcmI and NdeI sites and subsequently cloned into the HIV-rtTA molecular clone as BamHI-BglI fragments. All constructs were verified by sequence analysis.
rtTA activity assay. pLTR-2ΔtetO-luc expresses firefly luciferase from the LTR-2ΔtetO promoter derived from the HIV-rtTA molecular clone (Marzio et al, 2001; Marzio et al, 2002). pCMV-7tetO-luc, previously named pUHC13-3 (Gossen & Bujard, 1992), contains seven tetO elements located upstream of a minimal CMV promoter and the firefly luciferase gene. The plasmid pRL-CMV (Promega), in which the expression of Renilla luciferase is controlled by the CMV promoter, was used as an internal control to allow correction for differences in transfection efficiency. HeLa X1/6 cells are derived from the HeLa cervix carcinoma cell line and harbor chromosomally integrated copies of the CMV-7tetO firefly luciferase reporter construct (Baron et al, 1997). HeLa X1/6 and C33A cervix carcinoma cells (ATCC HTB31) (Auersperg, 1964) were cultured at 37° C. and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, minimal essential medium nonessential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin.
C33A and HeLa X1/6 cells were grown in 2-cm2 wells to 60% confluency and transfected by the calcium phosphate precipitation method. 1 μg of DNA mixture in 15 μl water was mixed with 25 μl of 50 mM HEPES (pH 7.1)-250 mM NaCl-1.5 mM Na2HPO4 and 10 μl of 0.6 M CaCl2, incubated at room temperature for 20 min and added to the culture medium. The DNA mixture consisted of 0.4 ng pCMV-rtTA, 20 ng pLTR-2ΔtetO-luc or pCMV-7tetO-luc, 0.5 ng pRL-CMV, and 980 ng pBluescript as carrier DNA for C33A cells, or 8 ng pCMV-rtTA, 2.5 ng PRL-CMV, and 990 ng pBluescript for HeLa X1/6 cells. Transfected cells were cultured for 20 hours at different dox concentrations, washed with DMEM, and subsequently cultured for 24 hours with fresh medium containing dox (the same concentrations as before the wash step). Cells were then lysed in Passive Lysis Buffer (Promega), and firefly and Renilla luciferase activities were determined with the Dual-Luciferase Reporter Assay (Promega) using a GloMax microplate luminometer (Promega). The expression of firefly and Renilla luciferase was within the linear range and no squelching effects were observed. The activity of the rtTA variants was calculated as the ratio of the firefly and Renilla luciferase activities, and corrected for between-session variation.
Evolution of HIV-rtTA after transient dox administration. To test the genetic stability of HIV-rtTA upon removal of the effector dox, we started 12 independent virus cultures in SupT1 T cells with dox (
Similar results were obtained with HIV-rtTAV9I G138D, an improved HIV-rtTA variant with two rtTA mutations (V9I and G138D) (example 1). The evolved viruses started to replicate without dox in 10 of the 12 cultures (
P56S mutation causes a tTA-like phenotype. The repeated selection of the P56S mutation in multiple, independent cultures strongly suggests its linkage to the observed loss of dox-control. To demonstrate that this amino acid substitution is indeed responsible for an altered rtTA phenotype, we cloned the P56S-mutated rtTA gene into the expression plasmid pCMV-rtTA and assayed its activity in a regular Tet-on system. The rtTA expression plasmid was transfected into C33A cells together with a reporter plasmid in which luciferase expression is controlled by the viral LTR-2ΔtetO promoter (Marzio et al, 2001; Marzio et al, 2002). Transfected cells were cultured for two days at different dox concentrations. We subsequently determined the intracellular luciferase level, which reflects rtTA activity (
We also analyzed rtTA activity in C33A cells transfected with a luciferase reporter under the control of a minimal CMV promoter coupled to an array of seven tetO elements (Gossen & Bujard, 1992), and in HeLa X1/6 cells that contain stably integrated copies of this CMV-7tetO luciferase construct (Baron et al, 1997). In both assays, we observed similar results as with the viral LTR-2ΔtetO promoter construct (
HIV-rtTAG19F E37L can lose dox-control by a P56S mutation. We have previously constructed an HIV-rtTA variant with the safety-lock mutations G19F and E37L that prevent the virus from losing dox-control during long-term culturing with dox (example 2). We now tested the stability of HIV-rtTAG19F E37L in the dox-washout experiment. This virus did lose dox-control in only one of the 12 cultures, and all other cultures did not show any replication in the absence of dox (
Safety-lock mutation at position 56. The P56S mutation is caused by a single nucleotide substitution (CCA to UCA). Such single nucleotide transitions (pyrimidine-pyrimidine or purine-purine substitutions) occur at a much higher frequency than single nucleotide transversions (pyrimidine-purine substitutions) or multiple nucleotide changes during HIV-1 reverse transcription (Berkhout et al, 2001; Berkhout & de Ronde, 2004). This mutational bias strongly influences the course of virus evolution (Keulen et al, 1996; Keulen et al, 1997). Accordingly, the undesired evolutionary route at position 56 is blocked by introducing alternative amino acid codons that require multiple nucleotide changes for HIV-rtTA to lose dox-control. In fact, we have successfully blocked the escape routes at positions 19 and 37 by such safety-lock mutations, which demonstrate the effectiveness of this strategy (example 2). To block all three observed escape routes of HIV-rtTA at the same time, the position 56 safety-lock mutation is ideally combined with the positions 19 and 37 mutations. To identify suitable amino acid substitutions, we made rtTA expression plasmids with all possible amino acids at position 56 in combination with the G19F and E37L mutations, and assayed their activity in HeLa X1/6 cells.
The activity of these 20 rtTA variants varies considerably (
In the codon table, every change in row or column represents a single nucleotide substitution. Apparently, the only position 56 codon that preserves dox-dependence (light grey) and requires more than a single nucleotide mutation to be converted to a codon that allows replication without dox (dark grey) is the AUA codon encoding an Isoleucine. However, the activity of the I variant at 1000 ng/ml dox is only 1% of the wild-type level (
Blocking loss of dox-control by triple safety-lock rtTA variant. We constructed HIV-rtTA molecular clones carrying triple safety-lock mutations G19F, E37L and P56K or P56Q, and tested their replication in SupT1 T cells with and without dox (
Our virus evolution experiments demonstrate that HIV-rtTA is at risk of escaping from dox-control by an amino acid substitution in rtTA at position 19, 37 or 56. To generate a safe HIV-rtTA virus, all three evolutionary routes are preferably blocked. We have previously blocked the position 19 and 37 routes by safety-lock mutations (e.g. G19F and E37L) that require multiple nucleotide changes to lose dox-control (example 2). We here demonstrate that the position 56 escape route is efficiently blocked by the introduction of an alternative amino acid at position 56 (e.g, P56K or P56Q) that requires at least one nucleotide transversion to convert rtTA into a dox-independent variant.
a The wild-type rtTA was previously described as rtTA2S-S2 (Urlinger et al. 2000).
b All variants (V1-V18) contain the F86Y (in the TetR domain) and A209T (in the VP16 activation domain) mutations.
Schematic of the HIV-rtTA genome. The inactivated Tat-TAR elements (crossed boxes) and the introduced rtTA-tetO elements are indicated. rtTA is a fusion protein of the E. coli Tet repressor (TetR) and the VP16 activation domain (AD) of herpes simplex virus. TetR contains a DNA-binding domain (DNA BD) (residues 1-44) and a regulatory core domain (residues 75-207) with a dimerization surface. (B) Flow-chart of the 24-well evolution experiment. Further details are provided in the text. (C) Gradual loss of dox-control in HIV-rtTA, HIV-rtTA 2ΔtetO (carrying the improved 2ΔtetO promoter configuration (Marzio et al. 2001; Marzio et al. 2002) and HIV-rtTAF86Y A209T (carrying the LTR-2ΔtetO promoter and the improved rtTAF86Y A209T gene (Das et al. 2004a). The HIV-rtTAG19F E37L variant developed in this study does not escape from dox-control. Plotted is the number of dox-dependent cultures as a function of the culture time. Each experiment was started with 24 independent cultures. (D) Amino acid substitutions observed in HIV-rtTA cultures that lost dox-control. In all cases, the G19E substitution resulted from a GGA to GAA codon mutation and the E37K substitution from a GAG to AAG mutation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05077623.6 | Nov 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2006/000575 | 11/17/2006 | WO | 00 | 10/21/2009 |
