BACKGROUND
an array of different viral transduction systems are being used currently in pre-clinical and clinical trials  <cit> .
among these, vectors based on the replication-defective adeno-associated virus type  <dig> have attracted special attention as tools for clinical gene transfer.
different characteristics, such as  the ability to transduce dividing as well as non-dividing cells,  high transduction rates in a wide range of tissues, and notably,  the unique safety properties, make aavs a promising vector in gene therapy initiatives  <cit> .
over the past few years, extensive studies have been carried out on different systems to regulate transgenes with small-molecule stimuli, preferentially clinically licensed agents.
started by the tetraycline-responsive tet system  <cit> , numerous other control modalities have followed including those responsive to streptogramin  <cit> , macrolide  <cit>  and aminocoumarines  <cit> , immunosuppressive agents   <cit> , hormones  <cit> , or susceptible to temperature  <cit> , quorum sensing molecules  <cit>  and gaseous acetaldehyde  <cit> .
to date, most of the experimental work with aav vectors in the gene-therapy field has focused on  the expression of therapeutic transgenes driven by strong constitutive promoters  <cit> ,  the regulated expression of transgenes based on the tetracycline-responsive teton and tetoff systems  <cit>  and, to lesser extent,  the regulated expression of transgenes by rapamycin-controlled transgene expression  <cit> .
until recently, in-vivo studies using recombinant aav particles have been limited by the production of high-titer and helper virus-free preparations.
however, with the development of helper-free production methods  <cit>  and improved purification and concentration protocols  <cit> , high-titer production of aav particles in the absence of helper-virus contamination was achieved, thereby opening the way to in vivo and clinical studies with aav-derived particles.
we report the design and validation of different aav type 2-based expression vectors, which enable macrolide-controlled transgene expression capitalizing on the recently developed erythromycin-responsive expression technology   <cit> .
e.rex systems exist in two different configurations:  the eoff arrangement consisting of a macrolide-dependent transactivator  repressor protein  and the herpes simplex virus vp <dig> transactivation domain) which binds and activates chimeric promoters  assembled by placing et-specific operator modules 5' of a minimal eukaryotic promoter in a macrolide-responsive manner.
since the presence of erythromycin turns transgene expression off by abolishing the et-petr interaction these control modalities are known as off-type or eoff systems  <cit> .
the eon technology consists of a macrolide-dependent transrepressor ,  <cit> ), a fusion of the e. coli mphr repressor protein  and the krab  transsilencing domain of the human kox- <dig> gene), which binds and represses chimeric promoters  assembled by placing e-krab-specific operator modules 3' of a constitutive eukaryotic promoter in a macrolide-inducible manner.
since the presence of erythromycin turns transgene expression on by releasing e-krab from petron, these control arrangements are known as on-type or eon systems  <cit> .
we have designed a set of recombinant aav vectors harboring eoff or eon-controlled expression units  on two independent vectors ,  on a single vector expressing the transgene and the transactivator in a dicistronic or bidirectional configuration and  on a single vector containing a constitutive promoter, driving transactivator expression, and the regulatable petr promoter, driving the gene of interest.
we have optimized performance, delivery and regulation, analyzed key characteristics such as reversibility and adjustability of the systems and validated the results with quantitative in-vitro as well as mouse studies.
RESULTS
design of recombinant adeno-associated viral particles for transduction of eoff-controlled transgene expression
we have engineered serotype 2-based adeno-associated viral particles for transduction of macrolide-responsive expression of the enhanced yellow fluorescent protein .
the generic design consisted of a set of two vectors: pdf <dig>  harboring an itr -flanked phcmv-  driven and pahgh-  terminated et <dig>  expression unit containing a β-globin intron  reported to increase transcript processing and overall et <dig> production levels and pdf <dig>  containing an itr- flanked expression unit encoding petr-  driven and pasv40-  terminated eyfp  expression cassette .
transgenic aav-derived particles produced by transient co-transfection of either pdf <dig>  pdf <dig> or pdf <dig> with the helper construct pdg providing constitutive processing and assembly functions  in trans into hek293-t were validated for regulated eyfp expression by co-transduction of pdf51- and pdf54-derived aav particles into representative cell types such as chinese hamster ovary cells , human fibrosarcoma cells , primary normal human dermal fibroblasts  and human breast cancer cells  cultivated in the presence  or absence  of the macrolide antibiotic erythromycin .
fluorescence micrographs of transduced populations maintained for  <dig> h in erythromycin-free medium revealed bright green fluorescence in all cell types.
transduced populations cultivated for two days in the presence of  <dig> μg/ml erythromycin showed no important eyfp expression.
as a positive control all cell types were transduced with aav particles harboring eyfp driven by a constitutive human cytomegolovirus immediate early promoter  .
one vector-based macrolide-responsive aav expression vectors
in order to enable delivery of macrolide-responsive transgene expression in a most compact format and a single transduction event we engineered expression of the macrolide-dependent transactivator et <dig> and the desired target gene into a single aav vector configuration.
the pioneering one-vector design concept consisted of tandem constitutive et <dig>  and macrolide-responsive eyfp expression units  placed between two itrs .
pdf141-  derived aav particles efficiently transduced ht- <dig> and mcf- <dig> and mediated high-level eyfp expression when transgenic populations were grown for  <dig> h in the absence of erythromycin .
to prevent any deregulation of petr by promoters or enhancers encoded in cis and to minimize the overall size of the transgene unit, we assembled  pdf <dig>  a dicistronic autoregulated petr-driven aav expression configuration harboring an itr-flanked petr-driven dicistronic expression cassette with eyfp in the first and et <dig> in the second cistron separated by a internal ribosome entry site of encephalomyocarditisviral origin  and  pdf <dig>  containing a bidirectional expression unit consisting of a central et1-specific operator etr flanked by petr driving eyfp expression in one direction and a phsp70min  driving et <dig> in the opposite direction.
following transduction of pdf124-derived aav particles, active petr produces a single transcript from which eyfp is translated in a classic cap-dependent manner whereas et <dig> production depends on cap-independent iresemcv-mediated translation initiation.
undetectable petr-mediated transcripts result in few initial et <dig> proteins, which, in the absence of erythromycin trigger auto-regulated high-level expression of this transactivator along with the cocistronically encoded eyfp.
in the presence of erythromycin, et <dig> originating from basal petr activity is inactivated, which interrupts the auto-regulated feed-forward transcription and results in repression of eyfp production.
after transduction of pdf89-derived aav particles, leaky et <dig> transcript initiate an auto-regulated expression circuit resulting in simultaneous expression of et <dig> and eyfp until et1-etr binding is abolished by erythromycin.
all three configurations yielded aav particles, which displayed good regulation performance upon trandsduction of ht- <dig> and mcf- <dig> and cultivation in the absence or in the presence of erythromycin .
while using equal genomic particle numbers the transduction efficiency varied between pdf <dig>  pdf <dig> and pdf <dig>  trandsuction rates of the bi-directional  and the dicistronic expression units  were significantly lower compared to the two-promoter set-up   which made facs-based quantitative analysis rather difficult .
however qualitative fluorescence microscopy suggested excellent regulation performance in individual transduced cells .
engineered aav-derived particles transducing tightly regulated production of secreted proteins
tight regulation of therapeutic transgenes remains a major challenge for current gene therapy initiatives.
in order to assess whether aav-based transduction systems enable delivery of tightly regulated expression of a human model glycoprotein, we constructed pdf <dig> harboring a petr-driven seap   .
co-transduction of ht- <dig> and mcf- <dig> with pdf77- and pdf51-  derived aav particles provided excellent regulation performance characterized by high-level seap production in the absence of erythromycin and repressed seap levels in the presence of erythromycin .
furthermore, we have designed and evaluated pdf <dig> , a one-vector expression configuration which is isogenic to pdf <dig> but encoding seap instead of eyfp.
pdf <dig> exhibited similar seap regulation profiles in ht- <dig> and mcf- <dig> compared to co-transduction of pdf77-/pdf51-derived aav particles suggesting that there is no interference between et1-driving psv <dig> and et1-specific petr .
overall, maximum expression levels for the one-vector configuration were even higher than for the binary setting likely because for the binary system two different particles have to transduce a single cell which is a more rare event.
transduction of ht- <dig> and mcf- <dig> with a constitutive control vector, pdf <dig>  showed increased seap production compared to fully induced pdf143-derived particles, which might be associated with either higher promoter strenght or the β-globin intron.
aavs engineered for eon-controlled transgene expression
various therapeutic initiatives require transgene control systems, which trigger heterologous target genes after administration of the regulating agent and remain repressed in the absence of the inducer  <cit> .
we have constructed binary eon-controlled aav-based expression systems consisting of pdf <dig> , and pdf <dig> , pdf <dig>  or pdf <dig> , which harbor eyfp under control of different e-krab-specific macrolide-inducible promoters characterized by different etr tandem repeats placed 3' of the constitutive psv <dig> promoter  <cit> .
functionality of the on-type system was validated by co-transduction of pdf126/pdf209-derived into ht- <dig> and mcf- <dig> and profiling of eyfp expression after  <dig> h cultivation in the presence or absence of erythromycin .
facs-based comparison of ht- <dig> co-transduced with pdf126-/pdf207-, pdf126-/pdf208- and pdf126-/pdf209-derived aav particles and cultivated for  <dig> h in the presence and absence of erythromycin revealed that although overall induction factors were almost identical among different particle combinations, basal and maximum expression levels were a function of the number of etr repeats associated with psv <dig> .
reversibility and adjustability of aav-transduced macrolide-controlled transgene expression
reversibility and adjustability are key characteristics for future clinical implementation of human-compatible transgene control modalities.
in order to assess the reversibility of macrolide-responsive transgene expression in an aav expression configuration we transduced ht- <dig> and mcf- <dig> with pdf143-derived aav particles and cultivated  in the presence  or absence  of em over  <dig> days,  in the presence  or absence  of em over  <dig> days and then incubated in reversed em conditions for the remaining three days and  in medium whose em status was alternated every three days from +em to -em to +em  or from -em to +em to -em .
seap accumulation was always measured prior to any em status switch.
ht- <dig> and mcf- <dig> showed excellent switching characteristics upon application of removal of erythromycin
in order to assess the dose-response characteristics of binary and one-vector-based macrolide-responsive aav transduction systems ht- <dig> and mcf- <dig> were transduced with pdf143- or co-transduced with pdf77-/pdf51-derived aav particles and cultivated for  <dig> h in the presence of different em concentrations before seap production was quantified.
whereas seap expression was completely repressed between  <dig> and  <dig> ng/ml for both configurations gradual decrease in antibiotic treatment resulted in dose-dependent increase of transgene expression until maximum expression levels were reached in the absence of em .
the differences in the dose-response characteristics of one-vector and binary configurations may result from different et1-petr stoichiometries associated with those technologies.
transgenic aav-derived particles mediate eoff-controlled transgene expression in mice
for in-vivo validation of aav-based macrolide-responsive transgene transduction we injected 5* <dig> pdf143-derived genomic aav particles intramuscularly into mice and treated them optionally with intraperitoneal erythromycin injections .
profiling of serum seap levels of treated mice showed significant production of this glycoprotein in mice, which had not received em doses whereas seap production was repressed whenever em was administered .
discussion
in recent years, gene delivery systems based on adeno-associated viral vectors have become one of the most promising tools for delivering transgenes in vivo.
after major setbacks in trials with retroviral as well as adenoviral vectors  <cit> , aavs present a strong alternative for the safe and efficient delivery of transgenes.
we have pioneered the delivery and regulation of transgenes by integrating the recently developed erythromycin-controllable e.rex system into aav type 2-derived backbones.
considering that erythromycin is a clinically licensed drug and that aav vectors have an excellent safety profile record, such a system is suitable for in vivo applications and is promising for clinical initiatives.
maximum erythromycin doses of up to  <dig> g/day in adults are in accordance with fda and ahfs guidelines  <cit> .
these doses are within the range of the amounts we have tested in mice.
we are therefore confident that the aav-encoded and erythromycin-controlled transgenes will be compatible with future clinical applications.
research, in which aav vectors deliver adjustable transgenes, has been conducted mainly with tetracycline responsive systems, which trigger or repress expression upon the addition of tetracycline  <cit> .
we have designed a range of aav type 2-based vectors encoding different configurations of the eoff expression system to evaluate optimal arrangements for further in vivo studies.
a binary set-up, where the transactivator is delivered on one vector and the gene of interest is driven by petr on another vector, worked well in vitro.
the expression levels, vector titers and regulation performance of the tested configurations were excellent, indicating an efficient co-transduction of the two types of transgenic aav particles.
regardless of whether intracellular or secreted transgenes were expressed, the regulation factors of all the tested cell lines were between  <dig> and  <dig>  although transgene expression mediated by aavs engineered for macrolide-responsive transgenes expression were typically lower after induction compared to isogenic aav derivatives containing constitutive promoters the overall production levels were in the same order of magnitude in vitro.
isogenic eon systems also enabled excellent regulation performance but overall induction factors were typically lower, since cells exclusively transduced with the transgene-encoding aav particle exhibit constitutive expression.
therefore, to achieve optimized eon-controlled transgene expression from transgenic aavs arranged in a binary vector set, the transrepressor-encoding aav has to be administered in excess compared to the transgene-encoding counterpart or cell lines have to be used, which are particular susceptible to aav-based transduction.
generally, even though binary systems have the disadvantage that two vectors must enter the same cell to obtain regulated expression, this design is advantageous for some applications, namely when delivering large transgenes.
although binary systems are powerful tools, in a therapeutic setting it may be desirable to combine the regulation modules on a single vector.
the advantages of such a setting are:  only one virus must enter the target cell to obtain regulated expression of the transgene,  exact virus titration is easier and  lower virus doses are required for comparable transgene expression.
we have pioneered both self-regulated and auto-regulated macrolide-responsive recombinant adeno-associated viral vectors for tightly controlled transgene expression.
although auto-regulated expression units are more compact than self-regulated configurations we had difficulty achieving reasonably high transduction efficiencies and expression levels.
since qrt-pcr-based analysis of viral particles revealed identical number of encapsidated genomes among our aav portfolio, the lower transduction rates of auto-regulated aavs may result from their genetic configuration rather than virus assembly.
in contrast, self-regulated expression units, consisting of a strong constitutive promoter driving the expression of the transactivator as well as a macrolide-responsive promoter driving the desired transgene on the same vector, showed excellent performance in a variety of cell types.
the vector design using compact promoter and polyadenylation elements in a self-regulated configuration allows the integration of a transgene with a size of up to  <dig> bp which is sufficient for various clinically important transgenes .
we did not observe promoter interference in the tested cell types, although the strong constitutive psv <dig> promoter driving et <dig> and the erythromycin-responsive promoter were on the same construct.
we were successful in expressing intracellular as well as secreted transgenes in a highly controlled manner and showed precise titration of transgene expression upon addition of varying erythromycin concentrations as well as reversibility upon the addition or removal of the regulating agent.
after switching from a fully repressed to a fully induced state or vice versa a short lag phase was observed before the new expression state was reached.
this behaviour might be due to trace amounts of antibiotic remaining in the culture  or due to the degradation kinetics of transcripts and transactivators
one-vector configurations would be suitable for in vivo studies and would enable efficient targeting of a wide variety of cells by systemic administration or by local injection of the viral particles encoding the therapeutic transgene.
we exemplified the functionality of the system in vivo by administrating pdf143-derived aav particles intramuscularly to mice and analyzing transgene expression in the presence and absence of antibiotic for several days.
most current studies on regulated transgene expression are carried out using retroviral vectors  <cit> .
although these systems are powerful and allow the efficient long-term expression of transgenes, they have several drawbacks, such as  the necessity of retroviral vectors for integration into transcriptionally active regions, which may interfere with desired transgene control,  random integration, possibly triggering oncogene activation and  silencing of the integrated transgene upon integration into the host chromosome.
adeno-associated viral vectors on the contrary, remain mainly episomal after entering the target cell and, therefore, do not have aforementioned drawbacks, thus providing a powerful alternative for upcoming gene-therapy studies.
CONCLUSIONS
we have designed an array of novel aav type 2-based expression vectors, which enable safe and efficient transduction of mammalian cells for macrolide-adjustable transgene expression.
we have pioneered binary on- and off-type systems as well as compact bidirectional, auto-regulated and self-regulated one vector expression configurations for regulation of intracellular as well as secreted proteins in mammalian cells.
we have also engineered a compact self-regulated aav which demonstrated efficient transduction, expression and regulation of a reporter gene in mice.

 an array of different viral transduction systems are being used currently in pre-clinical and clinical trials. among these, vectors based on the replication-defective adeno-associated virus type have attracted special attention as tools for clinical gene transfer. different characteristics, such as the ability to transduce dividing as well as non-dividing cells, high transduction rates in a wide range of tissues, and notably, the unique safety properties, make aavs a promising vector in gene therapy initiatives. over the past few years, extensive studies have been carried out on different systems to regulate transgenes with small-molecule stimuli, preferentially clinically licensed agents. started by the tetraycline-responsive tet system, numerous other control modalities have followed including those responsive to streptogramin, macrolide and aminocoumarines, immunosuppressive agents, hormones, or susceptible to temperature, quorum sensing molecules and gaseous acetaldehyde. to date, most of the experimental work with aav vectors in the gene-therapy field has focused on the expression of therapeutic transgenes driven by strong constitutive promoters, the regulated expression of transgenes based on the tetracycline-responsive teton and tetoff systems and, to lesser extent, the regulated expression of transgenes by rapamycin-controlled transgene expression. until recently, in-vivo studies using recombinant aav particles have been limited by the production of high-titer and helper virus-free preparations. however, with the development of helper-free production methods and improved purification and concentration protocols, high-titer production of aav particles in the absence of helper-virus contamination was achieved, thereby opening the way to in vivo and clinical studies with aav-derived particles. we report the design and validation of different aav type 2-based expression vectors, which enable macrolide-controlled transgene expression capitalizing on the recently developed erythromycin-responsive expression technology. e.rex systems exist in two different configurations: the eoff arrangement consisting of a macrolide-dependent transactivator repressor protein and the herpes simplex virus vp transactivation domain) which binds and activates chimeric promoters assembled by placing et-specific operator modules 5' of a minimal eukaryotic promoter in a macrolide-responsive manner. since the presence of erythromycin turns transgene expression off by abolishing the et-petr interaction these control modalities are known as off-type or eoff systems. the eon technology consists of a macrolide-dependent transrepressor, ), a fusion of the e. coli mphr repressor protein and the krab transsilencing domain of the human kox- gene), which binds and represses chimeric promoters assembled by placing e-krab-specific operator modules 3' of a constitutive eukaryotic promoter in a macrolide-inducible manner. since the presence of erythromycin turns transgene expression on by releasing e-krab from petron, these control arrangements are known as on-type or eon systems. we have designed a set of recombinant aav vectors harboring eoff or eon-controlled expression units on two independent vectors, on a single vector expressing the transgene and the transactivator in a dicistronic or bidirectional configuration and on a single vector containing a constitutive promoter, driving transactivator expression, and the regulatable petr promoter, driving the gene of interest. we have optimized performance, delivery and regulation, analyzed key characteristics such as reversibility and adjustability of the systems and validated the results with quantitative in-vitro as well as mouse studies.  design of recombinant adeno-associated viral particles for transduction of eoff-controlled transgene expression we have engineered serotype 2-based adeno-associated viral particles for transduction of macrolide-responsive expression of the enhanced yellow fluorescent protein. the generic design consisted of a set of two vectors: pdf harboring an itr -flanked phcmv- driven and pahgh- terminated et expression unit containing a β-globin intron reported to increase transcript processing and overall et production levels and pdf containing an itr- flanked expression unit encoding petr- driven and pasv40- terminated eyfp expression cassette. transgenic aav-derived particles produced by transient co-transfection of either pdf pdf or pdf with the helper construct pdg providing constitutive processing and assembly functions in trans into hek293-t were validated for regulated eyfp expression by co-transduction of pdf51- and pdf54-derived aav particles into representative cell types such as chinese hamster ovary cells, human fibrosarcoma cells, primary normal human dermal fibroblasts and human breast cancer cells cultivated in the presence or absence of the macrolide antibiotic erythromycin. fluorescence micrographs of transduced populations maintained for h in erythromycin-free medium revealed bright green fluorescence in all cell types. transduced populations cultivated for two days in the presence of μg/ml erythromycin showed no important eyfp expression. as a positive control all cell types were transduced with aav particles harboring eyfp driven by a constitutive human cytomegolovirus immediate early promoter. one vector-based macrolide-responsive aav expression vectors in order to enable delivery of macrolide-responsive transgene expression in a most compact format and a single transduction event we engineered expression of the macrolide-dependent transactivator et and the desired target gene into a single aav vector configuration. the pioneering one-vector design concept consisted of tandem constitutive et and macrolide-responsive eyfp expression units placed between two itrs. pdf141- derived aav particles efficiently transduced ht- and mcf- and mediated high-level eyfp expression when transgenic populations were grown for h in the absence of erythromycin. to prevent any deregulation of petr by promoters or enhancers encoded in cis and to minimize the overall size of the transgene unit, we assembled pdf a dicistronic autoregulated petr-driven aav expression configuration harboring an itr-flanked petr-driven dicistronic expression cassette with eyfp in the first and et in the second cistron separated by a internal ribosome entry site of encephalomyocarditisviral origin and pdf containing a bidirectional expression unit consisting of a central et1-specific operator etr flanked by petr driving eyfp expression in one direction and a phsp70min driving et in the opposite direction. following transduction of pdf124-derived aav particles, active petr produces a single transcript from which eyfp is translated in a classic cap-dependent manner whereas et production depends on cap-independent iresemcv-mediated translation initiation. undetectable petr-mediated transcripts result in few initial et proteins, which, in the absence of erythromycin trigger auto-regulated high-level expression of this transactivator along with the cocistronically encoded eyfp. in the presence of erythromycin, et originating from basal petr activity is inactivated, which interrupts the auto-regulated feed-forward transcription and results in repression of eyfp production. after transduction of pdf89-derived aav particles, leaky et transcript initiate an auto-regulated expression circuit resulting in simultaneous expression of et and eyfp until et1-etr binding is abolished by erythromycin. all three configurations yielded aav particles, which displayed good regulation performance upon trandsduction of ht- and mcf- and cultivation in the absence or in the presence of erythromycin. while using equal genomic particle numbers the transduction efficiency varied between pdf pdf and pdf trandsuction rates of the bi-directional and the dicistronic expression units were significantly lower compared to the two-promoter set-up which made facs-based quantitative analysis rather difficult. however qualitative fluorescence microscopy suggested excellent regulation performance in individual transduced cells. engineered aav-derived particles transducing tightly regulated production of secreted proteins tight regulation of therapeutic transgenes remains a major challenge for current gene therapy initiatives. in order to assess whether aav-based transduction systems enable delivery of tightly regulated expression of a human model glycoprotein, we constructed pdf harboring a petr-driven seap. co-transduction of ht- and mcf- with pdf77- and pdf51- derived aav particles provided excellent regulation performance characterized by high-level seap production in the absence of erythromycin and repressed seap levels in the presence of erythromycin. furthermore, we have designed and evaluated pdf, a one-vector expression configuration which is isogenic to pdf but encoding seap instead of eyfp. pdf exhibited similar seap regulation profiles in ht- and mcf- compared to co-transduction of pdf77-/pdf51-derived aav particles suggesting that there is no interference between et1-driving psv and et1-specific petr. overall, maximum expression levels for the one-vector configuration were even higher than for the binary setting likely because for the binary system two different particles have to transduce a single cell which is a more rare event. transduction of ht- and mcf- with a constitutive control vector, pdf showed increased seap production compared to fully induced pdf143-derived particles, which might be associated with either higher promoter strenght or the β-globin intron. aavs engineered for eon-controlled transgene expression various therapeutic initiatives require transgene control systems, which trigger heterologous target genes after administration of the regulating agent and remain repressed in the absence of the inducer. we have constructed binary eon-controlled aav-based expression systems consisting of pdf, and pdf, pdf or pdf, which harbor eyfp under control of different e-krab-specific macrolide-inducible promoters characterized by different etr tandem repeats placed 3' of the constitutive psv promoter. functionality of the on-type system was validated by co-transduction of pdf126/pdf209-derived into ht- and mcf- and profiling of eyfp expression after h cultivation in the presence or absence of erythromycin. facs-based comparison of ht- co-transduced with pdf126-/pdf207-, pdf126-/pdf208- and pdf126-/pdf209-derived aav particles and cultivated for h in the presence and absence of erythromycin revealed that although overall induction factors were almost identical among different particle combinations, basal and maximum expression levels were a function of the number of etr repeats associated with psv. reversibility and adjustability of aav-transduced macrolide-controlled transgene expression reversibility and adjustability are key characteristics for future clinical implementation of human-compatible transgene control modalities. in order to assess the reversibility of macrolide-responsive transgene expression in an aav expression configuration we transduced ht- and mcf- with pdf143-derived aav particles and cultivated in the presence or absence of em over days, in the presence or absence of em over days and then incubated in reversed em conditions for the remaining three days and in medium whose em status was alternated every three days from +em to -em to +em or from -em to +em to -em. seap accumulation was always measured prior to any em status switch. ht- and mcf- showed excellent switching characteristics upon application of removal of erythromycin in order to assess the dose-response characteristics of binary and one-vector-based macrolide-responsive aav transduction systems ht- and mcf- were transduced with pdf143- or co-transduced with pdf77-/pdf51-derived aav particles and cultivated for h in the presence of different em concentrations before seap production was quantified. whereas seap expression was completely repressed between and ng/ml for both configurations gradual decrease in antibiotic treatment resulted in dose-dependent increase of transgene expression until maximum expression levels were reached in the absence of em. the differences in the dose-response characteristics of one-vector and binary configurations may result from different et1-petr stoichiometries associated with those technologies. transgenic aav-derived particles mediate eoff-controlled transgene expression in mice for in-vivo validation of aav-based macrolide-responsive transgene transduction we injected 5* pdf143-derived genomic aav particles intramuscularly into mice and treated them optionally with intraperitoneal erythromycin injections. profiling of serum seap levels of treated mice showed significant production of this glycoprotein in mice, which had not received em doses whereas seap production was repressed whenever em was administered. discussion in recent years, gene delivery systems based on adeno-associated viral vectors have become one of the most promising tools for delivering transgenes in vivo. after major setbacks in trials with retroviral as well as adenoviral vectors, aavs present a strong alternative for the safe and efficient delivery of transgenes. we have pioneered the delivery and regulation of transgenes by integrating the recently developed erythromycin-controllable e.rex system into aav type 2-derived backbones. considering that erythromycin is a clinically licensed drug and that aav vectors have an excellent safety profile record, such a system is suitable for in vivo applications and is promising for clinical initiatives. maximum erythromycin doses of up to g/day in adults are in accordance with fda and ahfs guidelines. these doses are within the range of the amounts we have tested in mice. we are therefore confident that the aav-encoded and erythromycin-controlled transgenes will be compatible with future clinical applications. research, in which aav vectors deliver adjustable transgenes, has been conducted mainly with tetracycline responsive systems, which trigger or repress expression upon the addition of tetracycline. we have designed a range of aav type 2-based vectors encoding different configurations of the eoff expression system to evaluate optimal arrangements for further in vivo studies. a binary set-up, where the transactivator is delivered on one vector and the gene of interest is driven by petr on another vector, worked well in vitro. the expression levels, vector titers and regulation performance of the tested configurations were excellent, indicating an efficient co-transduction of the two types of transgenic aav particles. regardless of whether intracellular or secreted transgenes were expressed, the regulation factors of all the tested cell lines were between and although transgene expression mediated by aavs engineered for macrolide-responsive transgenes expression were typically lower after induction compared to isogenic aav derivatives containing constitutive promoters the overall production levels were in the same order of magnitude in vitro. isogenic eon systems also enabled excellent regulation performance but overall induction factors were typically lower, since cells exclusively transduced with the transgene-encoding aav particle exhibit constitutive expression. therefore, to achieve optimized eon-controlled transgene expression from transgenic aavs arranged in a binary vector set, the transrepressor-encoding aav has to be administered in excess compared to the transgene-encoding counterpart or cell lines have to be used, which are particular susceptible to aav-based transduction. generally, even though binary systems have the disadvantage that two vectors must enter the same cell to obtain regulated expression, this design is advantageous for some applications, namely when delivering large transgenes. although binary systems are powerful tools, in a therapeutic setting it may be desirable to combine the regulation modules on a single vector. the advantages of such a setting are: only one virus must enter the target cell to obtain regulated expression of the transgene, exact virus titration is easier and lower virus doses are required for comparable transgene expression. we have pioneered both self-regulated and auto-regulated macrolide-responsive recombinant adeno-associated viral vectors for tightly controlled transgene expression. although auto-regulated expression units are more compact than self-regulated configurations we had difficulty achieving reasonably high transduction efficiencies and expression levels. since qrt-pcr-based analysis of viral particles revealed identical number of encapsidated genomes among our aav portfolio, the lower transduction rates of auto-regulated aavs may result from their genetic configuration rather than virus assembly. in contrast, self-regulated expression units, consisting of a strong constitutive promoter driving the expression of the transactivator as well as a macrolide-responsive promoter driving the desired transgene on the same vector, showed excellent performance in a variety of cell types. the vector design using compact promoter and polyadenylation elements in a self-regulated configuration allows the integration of a transgene with a size of up to bp which is sufficient for various clinically important transgenes. we did not observe promoter interference in the tested cell types, although the strong constitutive psv promoter driving et and the erythromycin-responsive promoter were on the same construct. we were successful in expressing intracellular as well as secreted transgenes in a highly controlled manner and showed precise titration of transgene expression upon addition of varying erythromycin concentrations as well as reversibility upon the addition or removal of the regulating agent. after switching from a fully repressed to a fully induced state or vice versa a short lag phase was observed before the new expression state was reached. this behaviour might be due to trace amounts of antibiotic remaining in the culture or due to the degradation kinetics of transcripts and transactivators one-vector configurations would be suitable for in vivo studies and would enable efficient targeting of a wide variety of cells by systemic administration or by local injection of the viral particles encoding the therapeutic transgene. we exemplified the functionality of the system in vivo by administrating pdf143-derived aav particles intramuscularly to mice and analyzing transgene expression in the presence and absence of antibiotic for several days. most current studies on regulated transgene expression are carried out using retroviral vectors. although these systems are powerful and allow the efficient long-term expression of transgenes, they have several drawbacks, such as the necessity of retroviral vectors for integration into transcriptionally active regions, which may interfere with desired transgene control, random integration, possibly triggering oncogene activation and silencing of the integrated transgene upon integration into the host chromosome. adeno-associated viral vectors on the contrary, remain mainly episomal after entering the target cell and, therefore, do not have aforementioned drawbacks, thus providing a powerful alternative for upcoming gene-therapy studies.  we have designed an array of novel aav type 2-based expression vectors, which enable safe and efficient transduction of mammalian cells for macrolide-adjustable transgene expression. we have pioneered binary on- and off-type systems as well as compact bidirectional, auto-regulated and self-regulated one vector expression configurations for regulation of intracellular as well as secreted proteins in mammalian cells. we have also engineered a compact self-regulated aav which demonstrated efficient transduction, expression and regulation of a reporter gene in mice. 
capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice. moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. viral vectors, based on the adeno-associated virus type have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. we designed recombinant adeno-associated virus vectors for the regulated expression of transgenes in different configurations. to validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.
 adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. viral vectors, based on the adeno-associated virus type have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features.  we designed recombinant adeno-associated virus vectors for the regulated expression of transgenes in different configurations. we integrated the macrolide-responsive e.rex systems into raav backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. we tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types. moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. to validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.  capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice. 