Team:Heidelberg/Project/Mouse Infection

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==Introduction==
==Introduction==
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Gene therapy offers a great tool for treatment of various diseases such as failure, muscular dystrophies, and cystic fibrosis (Zincarelli, Soltys et al. 2008). <br>
+
Gene therapy offers a fantastic and unique tool for efficient treatment of various human diseases with a genetic cause, ranging from metabolic disorders and cancers to infections with different viral pathogens (Zincarelli, Soltys et al. 2008).  In principal, the underlying concept comprises the initial identification of a certain genetic defect, such as a deletion of a critical tumor suppressor gene which then causes cancer, or a mutation in a gene that is essential for a given metabolic pathway and whose loss or malfunction accordingly triggers symptoms of a disease. Once identified, gene therapists then attempt to correct this specific defect by introducing a correct copy of the missing or mutated gene back into the affected cell, hoping that this newly delivered gene will behave just like the original that it replaces. For transfer of this therapeutic gene sequence, one can choose between physical carriers, such as liposomes or nanoparticles, or alternatively, many groups engineer naturally occurring viruses, such as Adenoviruses, Lentiviruses or Adeno-associated viruses (AAV) as gene delivery vehicles. The latter hold the particular advantage that viruses have evolved to potently and, at least in many cases, specifically transfer their genetic cargo to cells, implying that a recombinant therapeutic DNA (or RNA) replacing these genes will behave just the same. <br>
-
The iGEM project of the Heidelberg team 2010 is dealing with tuning gene expression and directing target specificity.  
+
 
-
Therefore AAV capsids are shuffled and under a stringent selection pressure the perfect tissue specific virus with a shuffled capsid gene should be picked. Even though AAVs are the perfect viruses in the context of gene therapy, as the vector is neither pathogenic nor toxic in cell culture and in vivo ([http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References Grimm, 2002]) it is still not possible to selectively choose an AAV serotype which are tissue specific. It is for example known that a lot of rational modifications of AAV capsid genes (by inserting peptides or introducing some point mutations) may lead to viruses which are really efficient in cell culture but would never work in vivo. Therefore the capsid shuffling introduces a perfect method for constructing AAV capsid genes which can be shuffled in any combination of choice. ([http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References Grimm et al., 2008]) <br>
+
However, as one can easily imagine from the complexity of gene expression networks in all cells, especially in those of higher organisms, this whole process of transferring a foreign gene into a cell and expressing it there is everything but trivial, and success is not necessarily guaranteed. In essence, there are several main challenges that still need to be overcome before gene therapy, or more generally gene transfer, in humans can become a routine application, namely, improvements in 1) specificity, 2) potency, 3) control and 4) related to all this, safety. Unsurprisingly, there have been a plethora of attempts in the past to tackle these challenges, and a great deal of success has been reported in the literature. What is curious about these findings and advances, however, is that the vast majority of them have merely focused on the recombinant DNA template itself and have tried to (and often managed to) improve its particular properties, such as stability or encapsidation into the shell of a recombinant virus for delivery to the target cell.<br>
-
The possibility of targeting and even tuning expression in a certain tissue or cell line by using miRNA binding sites leads to even fine-tuned and more specific expression of the GOI. The binding sites can be either constructed by the miBS designer or by rational design. They can be validated using the miMeasure plasmid or the miRtuning construct. After having characterized the binding sites of choice a specific and tuned expression cassette can be introduced into a viral construct and infected into mice. And this is exactly what we did:<br>
+
 
-
We rationally designed binding sites, cloned them into the miTuner kit and characterized them. We produced and own shuffled capsid library and after applying selection pressure one clone showed already very fast good infection efficiencies in culture. For this very reason we tested our shuffled capsid and also an AAV8 WT capsid packaging a Luciferase construct driven by the same promoter. This constructs tagged with different binding site combinations of choice in the 3’UTR were used for producing viral constructs for testing on- off and tuning properties of our system.
+
Still, these particular approaches to improve the therapeutic DNA have taught us an important lesson, namely, that for successful, specific and potent gene transfer into human cells, especially into human patients, DNA is not enough ! Instead, there are two additional levels that are at least equally important and that have therefore been in the center of our team's focus: 1) the carrier used for gene delivery and 2) the control of gene expression inside the cell. More specifically, we have decided to study and improve the above already mentioned Adeno-associated virus (a large family of over 100 naturally occurring different serotypes) as a gene delivery vehicle due to a number of outstanding advantages of this particular virus for use in cell and tissue engineering, including the complete lack of pathogenicity of the parental wildtype viruses plus their capability to infect a wide range of target cells and tissues ([http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References Grimm, 2002]). On the other hand, this broad tropism can also be a disadvantage in cases where one would like to target a very specific cell type and concurrently avoid off-target gene expression elsewhere in the body. Therefore, several research groups (including the one of our instructor Dr. Grimm) have very recently begun to devise novel methods to molecularly engineer synthetic "designer" AAVs based on naturally occurring capsids which ideally combine and enhance their best properties. For instance, it is now possible to create entire libraries of AAV capsids by inserting short targeting peptides into exposed regions of the virus surface, or alternatively, by randomly point mutating single amino acids in the capsid proteins. The latest and most potent approach is, however, to shuffle entire capsid genes by first fragmenting and subsequently re-assembling the parts into hybrid particles. Ideally, the resulting chimeras will combine the assets of all parental viruses, such as high potency in a given cell type coupled with evasion of neutralization by human anti-AAV antibodies ([http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References Grimm et al., 2008]). <br>
 +
 
 +
Regarding the second critical level, regulation of gene expression following DNA delivery, it would be particularly crucial in the context of many gene transfer/therapy applications to be able to fine-regulate the level of the resulting protein expression, considering that uncontrolled over-production of certain proteins can be highly toxic or even lethal to cells and tissues. Unfortunately, as mentioned above, most approaches in this direction thus far have mainly focused on the DNA itself and accordingly mostly dealt with the development of improved promoter sequences that mediate controlled gene expression under certain conditions. What has been largely ignored until very recently, however, is the fact that gene expression can also be regulated on the RNA level, namely, via RNAi technologies. In this respect, we reasoned that the novel and broadly applicable miRNA tools engineered by our iGEM team should prove extremely useful in combination with gene therapy vectors, considering that they have been devised to permit very tight control over the expression of a given gene on the RNA level (by controlling translation and/or mRNA stability, which is the natural mechanism of miRNA action). Specifically, we wanted to explore the feasibility to fuse a gene of interest (GOI) with binding sites for either a naturally occurring miRNA or for a synthetic miRNA that is co-expressed from a second "tuning" vector. In either case, the binding site can then be designed to match the mi/shRNA either perfectly, which will result in nearly complete knockdown of gene expression, or it can be imperfect, which will yield a more subtle reduction in gene expression (for details, see our synthetic miRNA kit).  <br>
 +
 
 +
Accordingly, we utilized our various novel tools to establish an entirely new system for fine-regulated gene expression from viral gene therapy vectors, in which control is exerted no longer on the DNA level, but in addition also on the steps of 1) gene delivery and 2) fine-tuning (Figure X).       <br>
 +
 
 +
Most importantly, as with any novel system for regulated gene expression, there is no guarantee that results obtained in cultured cells are relevant for, and will straight-forwardly translate into, identical finding in living animals or ultimately human patients. Therefore, we reasoned that it would be mandatory following the design and in vitro validation of our novel constructs to also assay and ideally confirm their functionality in living organisms in vivo, i.e., in adult mice.
 +
In the following, we will summarize our results from the in vivo evaluation of the two main strategies outlined above in the livers of adult mice and will demonstrate how modifying the viral gene vehicle capsid can drastically affect the specificity and level of gene expression in cultured cells versus living animals, and how engineering a given gene expression cassette by adding perfect or imperfect mi/shRNA binding sites can further fine-tune the strength of gene/protein expression at will.  
 +
<br>
==Results==
==Results==

Revision as of 03:47, 28 October 2010

in vivo study

Abstract

Gene therapy offers a great tool for treatment of various human diseases and has shown tremendous promise and success in many recent clinical trials. Nevertheless, two most critical hurdles that remain are 1) the development of delivery vectors that allow for the highly cell- and tissue-specific expression of a gene of interest (GOI), as well as 2) the implementation of novel systems that permit the tightly controlled and fine-tuned expression of this gene in human patients. To fill in these two essential gaps, the iGEM Team 2010 of Heidelberg is introducing molecular evolution of viral gene transfer vectors as well as mi(cro)RNA-based gene regulation technology into the realm of synthetic biology. By combining these two powerful new methodologies, we have succeeded in engineering synthetic viral miRNA vectors that we could demonstrate to provide potent, specific and regulated gene expression in cultured mouse and human hepatocytes. Most importantly, we also document the successful use of our novel approach to achieve tightly controlled and liver-specific expression of a vector-encoded recombinant DNA in intact livers of adult mice. Our work and data thus pave the way for the future further expansion and utilization of our unique miRNA/vector kits in numerous biotechnological and especially biomedical applications.

Introduction

Gene therapy offers a fantastic and unique tool for efficient treatment of various human diseases with a genetic cause, ranging from metabolic disorders and cancers to infections with different viral pathogens (Zincarelli, Soltys et al. 2008). In principal, the underlying concept comprises the initial identification of a certain genetic defect, such as a deletion of a critical tumor suppressor gene which then causes cancer, or a mutation in a gene that is essential for a given metabolic pathway and whose loss or malfunction accordingly triggers symptoms of a disease. Once identified, gene therapists then attempt to correct this specific defect by introducing a correct copy of the missing or mutated gene back into the affected cell, hoping that this newly delivered gene will behave just like the original that it replaces. For transfer of this therapeutic gene sequence, one can choose between physical carriers, such as liposomes or nanoparticles, or alternatively, many groups engineer naturally occurring viruses, such as Adenoviruses, Lentiviruses or Adeno-associated viruses (AAV) as gene delivery vehicles. The latter hold the particular advantage that viruses have evolved to potently and, at least in many cases, specifically transfer their genetic cargo to cells, implying that a recombinant therapeutic DNA (or RNA) replacing these genes will behave just the same.

However, as one can easily imagine from the complexity of gene expression networks in all cells, especially in those of higher organisms, this whole process of transferring a foreign gene into a cell and expressing it there is everything but trivial, and success is not necessarily guaranteed. In essence, there are several main challenges that still need to be overcome before gene therapy, or more generally gene transfer, in humans can become a routine application, namely, improvements in 1) specificity, 2) potency, 3) control and 4) related to all this, safety. Unsurprisingly, there have been a plethora of attempts in the past to tackle these challenges, and a great deal of success has been reported in the literature. What is curious about these findings and advances, however, is that the vast majority of them have merely focused on the recombinant DNA template itself and have tried to (and often managed to) improve its particular properties, such as stability or encapsidation into the shell of a recombinant virus for delivery to the target cell.

Still, these particular approaches to improve the therapeutic DNA have taught us an important lesson, namely, that for successful, specific and potent gene transfer into human cells, especially into human patients, DNA is not enough ! Instead, there are two additional levels that are at least equally important and that have therefore been in the center of our team's focus: 1) the carrier used for gene delivery and 2) the control of gene expression inside the cell. More specifically, we have decided to study and improve the above already mentioned Adeno-associated virus (a large family of over 100 naturally occurring different serotypes) as a gene delivery vehicle due to a number of outstanding advantages of this particular virus for use in cell and tissue engineering, including the complete lack of pathogenicity of the parental wildtype viruses plus their capability to infect a wide range of target cells and tissues (Grimm, 2002). On the other hand, this broad tropism can also be a disadvantage in cases where one would like to target a very specific cell type and concurrently avoid off-target gene expression elsewhere in the body. Therefore, several research groups (including the one of our instructor Dr. Grimm) have very recently begun to devise novel methods to molecularly engineer synthetic "designer" AAVs based on naturally occurring capsids which ideally combine and enhance their best properties. For instance, it is now possible to create entire libraries of AAV capsids by inserting short targeting peptides into exposed regions of the virus surface, or alternatively, by randomly point mutating single amino acids in the capsid proteins. The latest and most potent approach is, however, to shuffle entire capsid genes by first fragmenting and subsequently re-assembling the parts into hybrid particles. Ideally, the resulting chimeras will combine the assets of all parental viruses, such as high potency in a given cell type coupled with evasion of neutralization by human anti-AAV antibodies (Grimm et al., 2008).

Regarding the second critical level, regulation of gene expression following DNA delivery, it would be particularly crucial in the context of many gene transfer/therapy applications to be able to fine-regulate the level of the resulting protein expression, considering that uncontrolled over-production of certain proteins can be highly toxic or even lethal to cells and tissues. Unfortunately, as mentioned above, most approaches in this direction thus far have mainly focused on the DNA itself and accordingly mostly dealt with the development of improved promoter sequences that mediate controlled gene expression under certain conditions. What has been largely ignored until very recently, however, is the fact that gene expression can also be regulated on the RNA level, namely, via RNAi technologies. In this respect, we reasoned that the novel and broadly applicable miRNA tools engineered by our iGEM team should prove extremely useful in combination with gene therapy vectors, considering that they have been devised to permit very tight control over the expression of a given gene on the RNA level (by controlling translation and/or mRNA stability, which is the natural mechanism of miRNA action). Specifically, we wanted to explore the feasibility to fuse a gene of interest (GOI) with binding sites for either a naturally occurring miRNA or for a synthetic miRNA that is co-expressed from a second "tuning" vector. In either case, the binding site can then be designed to match the mi/shRNA either perfectly, which will result in nearly complete knockdown of gene expression, or it can be imperfect, which will yield a more subtle reduction in gene expression (for details, see our synthetic miRNA kit).

Accordingly, we utilized our various novel tools to establish an entirely new system for fine-regulated gene expression from viral gene therapy vectors, in which control is exerted no longer on the DNA level, but in addition also on the steps of 1) gene delivery and 2) fine-tuning (Figure X).

Most importantly, as with any novel system for regulated gene expression, there is no guarantee that results obtained in cultured cells are relevant for, and will straight-forwardly translate into, identical finding in living animals or ultimately human patients. Therefore, we reasoned that it would be mandatory following the design and in vitro validation of our novel constructs to also assay and ideally confirm their functionality in living organisms in vivo, i.e., in adult mice. In the following, we will summarize our results from the in vivo evaluation of the two main strategies outlined above in the livers of adult mice and will demonstrate how modifying the viral gene vehicle capsid can drastically affect the specificity and level of gene expression in cultured cells versus living animals, and how engineering a given gene expression cassette by adding perfect or imperfect mi/shRNA binding sites can further fine-tune the strength of gene/protein expression at will.

Results

Mice injection

The tail vein injection was chosen so as to assess AAV serotype tissue tropism; the luciferase transgene was used for visualizing the relative vector distribution in all the animals in a real-time manner. This allows for in vivo imaging and time lapse experiments.

Bioluminescence imaging







miR122 OFF-targetting


hAAT tuning
HeLa Transfection

Discussion

Methods

Contructs

The in vivo analysis should enlighten our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression. The following constructs have been subcloned separately into the AAV context to accomplish those tasks:

  1. positive control,
  2. off-targeting construct,
  3. synthetic tuning construct and
  4. on-targeting construct.


All but one virus were packaged by the AAV rep and cap gene with Adenovirus 5 (Ad5) as a helper plasmid. Accordingly, one virus construct was packaged into a shuffled cap gene from our homology based capsid shuffling attempt.

  1. The positive control (see sidebar, fig. 1) consisted of the SV40 promoter driving a firefly luciferase (luc2) gene, thereby leading to an unspecific expression of the luciferase protein in all mice tissues. In addition to packaging this construct into a wild type AAV virus, the positive control was also packaged as a transgene into our shuffled capsid which after random selection was already able to positively transduce Huh7 and HepG2 cells in vitro.
  2. The off-targeting construct (see sidebar, fig. 2) was composed of an SV40 promoter driving a firefly luciferase (luc2) gene with binding sites against miR-122 behind it. In order to achieve the highest expression in all mice cells but the liver cells - a single perfect binding site of miR-122 was used for in vivo study.
  3. The synthetic tuning construct (see sidebar, fig. 3) consisted of two viruses injected at the same time in the mice. The one virus packaged the expression construct of shRNA haat driven by the H1 promoter ("tuning" construct, see sidebar, fig.3). The second virus packaged the following transgene: SV40 promoter driving luc2 with shRNA haat binding site behind it ("tuned" construct, see sidebar, fig. 4). In order to ensure a synthetic tuning effect, a perfect binding site and one with a bulge that was introduced at position 9-12 were used for in vivo experiments, respectively. Those two binding sites should lead to a significant knockdown in the first case and a slight repression of luciferase expression in the latter as compared to the positive control.
  4. The on-targeting construct consisted of two independent viruses which were co-infected into mice, as well. One of these viruses packaged the Tet Repressor (TetR) driven by an SV40 promoter ("repressor" construct, see sidebar, fig. 5). The expression of TetR is under the control of miR-122 as four binding sites of this miRNA were cloned into the 3’UTR of the gene. The second virus was composed of an SV40 promoter driving the Tet operator (TetO2) which monitors the expression of luc2 ("operator" construct, see sidebar, fig. 6). With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is down-regulated by miRNA 122.

Production of recombinant virus

The viruses were produced in HEK 293-T cells and purified on an iodixanol gradient according to the virus production protocol.

Before infection, the titer of the viruses was quantified using quantitative realtime PCR.

Procedure involving animals

The mouse experiments were conducted in accordance with the animal facility of the German Cancer Research Center in Heidelberg. Female NMRI mice were obtained from a collaboration with Dr. Oliver Müller. At 8-10 weeks of age, the animals were injected in the tail vein (TV), with ~ 1x1011 particles of AAV-SV40-luciferase in 200µl of 1x phosphate-buffered saline. The mice are transferred to a holding device which restrains the mouse while allowing access to the tail vein. The tails were warmed before the injections and injections were carried out using 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity (Zincarelli et al., 2008).

in vivo animal imaging

Mice were anesthesized in an isofluran chamber. The mice were injected intraperitoneally with 200µl of a 30 mg/ml concentration of D-luciferin. This injection starts the luminescence of luc2. Mice were measured for one to seven minutes post injection under the in vivo bioluminometer.

References

Grimm, D. (2002). "Production methods for gene transfer vectors based on adeno-associated virus serotypes." Methods 28(2): 146-157.
Grimm, D., J. S. Lee, et al. (2008). "In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses." J Virol 82(12): 5887-5911.
Zincarelli, C., S. Soltys, et al. (2008). "Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection." Mol Ther 16(6): 1073-1080.