Current Opinion in Molecular Therapeutics, Sep 1, 2009
The clinical application of gene therapy has become a reality with the treatment of patients with... more The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.
The clinical application of gene therapy has become a reality with the treatment of patients with... more The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This revie...
Regenerative medicine using mesenchymal stem cells for the purposes of tissue repair has garnered... more Regenerative medicine using mesenchymal stem cells for the purposes of tissue repair has garnered considerable public attention due to the potential of returning tissues and organs to a normal, healthy state after injury or damage has occurred. To achieve this, progenitor cells such as pericytes and bone marrow-derived mesenchymal stem cells can be delivered exogenously, mobilised and recruited from within the body or transplanted in the form organs and tissues grown in the laboratory from stem cells. In this review, we summarise the recent evidence supporting the use of endogenously mobilised stem cell populations to enhance tissue repair along with the use of mesenchymal stem cells and pericytes in the development of engineered tissues. Finally, we conclude with an overview of currently available therapeutic options to manipulate endogenous stem cells to promote tissue repair.
Abstract Currently used vectors in human gene therapy suffer from a number of limitations with re... more Abstract Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a ...
Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeuti... more Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.
DNA vectors have the potential to become powerful medical tools for treatment of human disease. T... more DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.
Non-viral vectors have not been extensively investigated in neonatal mice due to the poor efficie... more Non-viral vectors have not been extensively investigated in neonatal mice due to the poor efficiency of the delivery methods available. Understanding the effects of non-viral vectors during early development is vital to develop safe gene therapy treatments where irreversible pathological processes may be avoided by early gene reconstitution. Here we describe a simple and effective method for the systemic administration of non-viral vectors via the superior temporal vein of mouse pups at 1.5 days of age. We show that injection of polyethylenimine (PEI)-complexed plasmid DNA (pDNA) intravenously results in effective transfection in the liver, lung, heart, spleen, brain and kidney. We also investigate the specific targeting of transgene expression to the proliferating neonate liver using a liver-specific plasmid containing a Scaffold Matrix Attachment Region (S/MAR) element, which has previously been shown to confer long-term expression in adult mouse liver. Using bioluminescent imaging, a gradual increase in transgene expression was observed which peaked at days 11-12, before the reduction of expression to background levels by day 25, suggestive of vector copy number loss. We conclude that non-viral vectors can successfully be used for systemic delivery to neonatal mice and that this technique is likely to open a host of early therapeutic possibilities for gene transfer by a range of non-viral vector formulations.
In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which ... more In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.
Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained trans... more Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose-dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas. This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases.
Recently, we reported for the first time the development of pH-triggered nanoparticles for the fu... more Recently, we reported for the first time the development of pH-triggered nanoparticles for the functional delivery of small interfering RNA (siRNA) to liver for treatment of hepatitis B virus infections in vivo. Here, we report on systematic formulation and biophysical studies of three different pH-triggered nanoparticle formulations looking for ways to improve on the capabilities of our previous nanoparticle system. We demonstrate how pH-triggered, PEGylated siRNA nanoparticles stable with respect to aggregation in 80% serum can still release siRNA payload at pH 5.5 within 30 min. This capability allows functional delivery to cultured murine hepatocyte cells in vitro, despite a high degree of PEGylation (5 mol %). We also demonstrate that pH-triggered, PEGylated siRNA nanoparticles typically enter cells by clathrin-coated pit endocytosis, but functional delivery requires membrane fusion events (fusogenicity). Biodistribution studies indicate that >70% of our administered nanoparticles are found in liver hepatocytes, post intravenous administration. Pharmacodynamic experiments show siRNA delivery to murine liver effecting maximum knockdown 48 h post administration from a single dose, while control (nontriggered) nanoparticles require 96 h and two doses to demonstrate the same effect. We also describe an anti-hepatitis C virus (HCV) proof-of-concept experiment indicating the possibility of RNAi therapy for HCV infections using pH-triggered, PEGylated siRNA nanoparticles.
Current Opinion in Molecular Therapeutics, Sep 1, 2009
The clinical application of gene therapy has become a reality with the treatment of patients with... more The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.
The clinical application of gene therapy has become a reality with the treatment of patients with... more The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This revie...
Regenerative medicine using mesenchymal stem cells for the purposes of tissue repair has garnered... more Regenerative medicine using mesenchymal stem cells for the purposes of tissue repair has garnered considerable public attention due to the potential of returning tissues and organs to a normal, healthy state after injury or damage has occurred. To achieve this, progenitor cells such as pericytes and bone marrow-derived mesenchymal stem cells can be delivered exogenously, mobilised and recruited from within the body or transplanted in the form organs and tissues grown in the laboratory from stem cells. In this review, we summarise the recent evidence supporting the use of endogenously mobilised stem cell populations to enhance tissue repair along with the use of mesenchymal stem cells and pericytes in the development of engineered tissues. Finally, we conclude with an overview of currently available therapeutic options to manipulate endogenous stem cells to promote tissue repair.
Abstract Currently used vectors in human gene therapy suffer from a number of limitations with re... more Abstract Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a ...
Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeuti... more Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.
DNA vectors have the potential to become powerful medical tools for treatment of human disease. T... more DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.
Non-viral vectors have not been extensively investigated in neonatal mice due to the poor efficie... more Non-viral vectors have not been extensively investigated in neonatal mice due to the poor efficiency of the delivery methods available. Understanding the effects of non-viral vectors during early development is vital to develop safe gene therapy treatments where irreversible pathological processes may be avoided by early gene reconstitution. Here we describe a simple and effective method for the systemic administration of non-viral vectors via the superior temporal vein of mouse pups at 1.5 days of age. We show that injection of polyethylenimine (PEI)-complexed plasmid DNA (pDNA) intravenously results in effective transfection in the liver, lung, heart, spleen, brain and kidney. We also investigate the specific targeting of transgene expression to the proliferating neonate liver using a liver-specific plasmid containing a Scaffold Matrix Attachment Region (S/MAR) element, which has previously been shown to confer long-term expression in adult mouse liver. Using bioluminescent imaging, a gradual increase in transgene expression was observed which peaked at days 11-12, before the reduction of expression to background levels by day 25, suggestive of vector copy number loss. We conclude that non-viral vectors can successfully be used for systemic delivery to neonatal mice and that this technique is likely to open a host of early therapeutic possibilities for gene transfer by a range of non-viral vector formulations.
In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which ... more In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.
Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained trans... more Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose-dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas. This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases.
Recently, we reported for the first time the development of pH-triggered nanoparticles for the fu... more Recently, we reported for the first time the development of pH-triggered nanoparticles for the functional delivery of small interfering RNA (siRNA) to liver for treatment of hepatitis B virus infections in vivo. Here, we report on systematic formulation and biophysical studies of three different pH-triggered nanoparticle formulations looking for ways to improve on the capabilities of our previous nanoparticle system. We demonstrate how pH-triggered, PEGylated siRNA nanoparticles stable with respect to aggregation in 80% serum can still release siRNA payload at pH 5.5 within 30 min. This capability allows functional delivery to cultured murine hepatocyte cells in vitro, despite a high degree of PEGylation (5 mol %). We also demonstrate that pH-triggered, PEGylated siRNA nanoparticles typically enter cells by clathrin-coated pit endocytosis, but functional delivery requires membrane fusion events (fusogenicity). Biodistribution studies indicate that >70% of our administered nanoparticles are found in liver hepatocytes, post intravenous administration. Pharmacodynamic experiments show siRNA delivery to murine liver effecting maximum knockdown 48 h post administration from a single dose, while control (nontriggered) nanoparticles require 96 h and two doses to demonstrate the same effect. We also describe an anti-hepatitis C virus (HCV) proof-of-concept experiment indicating the possibility of RNAi therapy for HCV infections using pH-triggered, PEGylated siRNA nanoparticles.
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