1. Introduction
Gene-replacement gene therapy has been under development for a number of years. In spite of the large amount of research invested into developing gene therapy for the treatment of recessive genetic disorders only a limited number of patients world-wide have received the benefits. In addition, several high profile adverse events in gene therapy trials have lead to an increasing awareness of the challenges facing gene therapy treatments before they become established in the clinic. This has necessitated the development of novel advances in gene therapy vector design and delivery. This chapter will focus on the development of gene expression vectors incorporating native genomic regulatory elements that ensure transgene expression is physiologically relevant. Three main advances will be discussed here in detail; the use of whole genomic DNA loci to ensure physiologically-regulated transgene expression; development of viral vectors based on the herpes simplex virus type 1 for delivery of whole genomic DNA loci; and the development of genomic mini-gene vectors that contain native regulatory regions for the physiologically-regulated expression of cDNA mini-genes.
The principal aim of gene-replacement gene therapy is to complement the loss of function of an endogenous gene by supplying an exogenous ‘working’ copy in
Historically, BAC vectors have been discounted for gene therapy purposes as there was no viral delivery system with the transgene capacity for a whole genomic locus which may be ≥100 kb. Recently, viral vectors based on the herpes simplex virus type (HSV-1) amplicons have been developed and shown to have a transgene capacity of over 100 kb and a broad cell tropism making them an attractive means of delivering large transgenes for the purposes of gene therapy. Currently HSV-1 amplicons have been used in a number of gene complementation studies which will be reviewed in detail here.
Recent work in our laboratory has adapted the genomic locus approach for the treatment of familial hypercholesterolaemia (FH). FH is a condition caused by mutations in the
2. Physiologically-relevant gene expression vectors: use of a complete genomic locus
The central aim of gene-replacement gene therapy is to complement the loss of function of an endogenous gene by supplying a working copy of that gene
2.1. Complete genomic locus ensures gene expression in the correct genomic context
There are several issues which may confound the use of cDNA expression cassettes to complement the loss of function of an endogenous gene: aberrant spatial expression dynamics resulting in gene expression in ‘off-target’ cells; aberrant temporal dynamics resulting in continuous expression of a transgene with possible cytotoxic consequences; transgene over-expression at supra-physiological levels; and, the inability to produce multiple splice variants.
Transgenic mice offer an interesting insight into the benefits of using native genomic loci over cDNA expression systems to investigate the function of genes. For some genes it is essential that they are expressed in the correct spatial, developmental and temporal context to ensure functionality. The β-globin gene cluster is an excellent example of this. This genomic locus consists of five separate genes (5’-ε-Gγ-Aγ-δ-β-3’) that are expressed at different developmental stages (Huang et al. 2000). The γ genes are expressed in foetal erythroid tissues while the δ and β are expressed in adult haematopoietic cells of the erythroid lineage. The expression of these genes in under the control of a region called the locus control region (Huang, Liu et al. 2000). In mice expressing the β-globin from a cDNA expression plasmid without the locus control region, low levels of protein are detected with no tissue-specificity (Magram et al. 1985; May et al. 2000; Vadolas et al. 2005). The use of the entire genomic locus of the β-globin gene cluster which included the locus control regions resulted in spatial and temporal expression profiles that mimicked the native profile (Porcu et al. 1997; Vadolas, Wardan et al. 2005).
Further examples of the advantages of using the complete genomic locus comes from mice lacking either the frataxin (
Other examples include comparisons between mice expressing the amyloid precursor protein (APP) as either a cDNA construct or as a complete locus within a YAC. The APP gene is involved with the development of Alzheimer’s disease. It is a complex genomic locus comprising 18 exons that are alternatively spliced to give rise to four distinct transcripts (Hsiao et al. 1996). Mice expressing the APP cDNA vector do not express APP protein in the correct genomic context limiting the relevance of biological information obtained from these animals (Lamb 1995; Lamb et al. 1997). Mice expressing APP from the YAC construct displayed physiologically-relevant APP protein expression making them a far superior tool for the study of how APP might contribute to the development of Alzheimer’s disease (Lamb, Call et al. 1997).
The advantages of using BAC plasmids to generate transgenic mice is now widely accepted. BAC transgenics have been shown, for example, to rescue knockout phenotypes in mice lacking the Pkd1 gene involved in polycystic kidney disease (Pritchard et al. 2000) and mice lacking β-globin genes (Vadolas et al. 2002; Jamsai et al. 2005; Vadolas, Wardan et al. 2005; Jamsai et al. 2006). BACs have also been useful in investigating novel genomic expression control regions. A negative regulatory region in the Wilson’s disease gene was characterised using BAC plasmids (Bochukova et al. 2003). BACs were also used to characterise the locus control regions responsible for the differential expression of Myf5 in skeletal muscle (Carvajal et al. 2001; Zammit et al. 2004). In addition to this, insertion of reporter genes into BAC plasmids has enabled the understanding of spatial and temporal expression dynamics of many genes such as Nkx2-5 (Chi et al. 2003). Recently BAC transgenesis has been used in studies of immunomodulation (Kulik et al. 2011), blood vessel development (Ishitobi et al. 2010) and in generating mouse models of Parkinson’s disease that more closely recapitulate deficits in the human disease (Li et al. 2009). These studies represent a small sub-section of the work being performed using whole genomic loci to better understand gene function. They demonstrate that the use of native regulatory regions can yield more biologically-relevant data than over-expression studies. This is important in the generation of mouse models of disease and also in the development of therapeutic protocols to treat genetic disease.
2.2. Complete genomic locus for therapy
Transgenic animals offer extensive evidence that the use of cDNA expression vectors often does not result in physiologically-relevant expression patterns. In terms of gene therapy the use of these cDNA vectors may not be appropriate for diseases where the correct physiological expression of the transgene is vital for therapeutic effect and to protect cells from ectopic or cytotoxic over-expression, where proteins expressed with no control result in pathological changes in the transduced cell.
The use of a complete genomic DNA region in the design of gene therapy vectors is still a relatively new field. Manipulation and use of such large pieces of DNA can be challenging. Success has been seen however with a range of genes using a number of different techniques to isolate and deliver the locus.
Alternatives to BAC plasmids for delivery of large genomic inserts are also being investigated. Human artificial chromosomes (HACs) for example offer advantages over the bacterial counterparts. HAC vectors are able to replicate and segregate without integration into the host-cell chromosomes and are capable of carrying very large amounts of DNA. HACs have been shown to be an effective means of generating transgenic mice (Suzuki 2006). They have also been used to express
Viral vectors have also been developed to achieve infectious delivery of large genomic sequences. A gutless adenovirus with a transgene capacity of 36 kb was used to deliver the whole locus of the human α1 antitrypsin gene (
2.3. Infectious delivery of a complete genomic locus – HSV-1 amplicons
The large size of a compete genomic locus precludes their use with most viral vector systems which typically have a transgene capacity of less than 20 kb. Vectors based on the herpes virus family however have a much larger transgene capacity. HSV-1 in particular is well described and widely used. Wild-type HSV-1 infects mucosa and establishes a latent phase in sensory neurons. HSV-1 infection produces cold sores in symptomatic infected individuals and 90% of the population has circulating antibodies (Corey and Spear 1986; Bowers et al. 2003). The HSV-1 genome consists of 152 kb of double stranded DNA. Of this only two non-coding regions are required for the packaging of DNA plasmids into HSV-1 virions (Spaete and Frenkel 1982; Spaete and Frenkel 1985). Inclusion of these two packaging signals, the
HSV-1 amplicons are capable of infecting dividing and non-dividing cells including, but not limited to; neurons, such as those of the dorsal root ganglion (Marsh et al. 2000), thalamus (Costantini et al. 1999), cortex (Agudo et al. 2002), hippocampus (Adrover et al. 2003), glial cells (Marsh, Dekaban et al. 2000), gliomas (Shah et al. 2004), skeletal muscle (Wang et al. 2000; Wang et al. 2002) and osteoblasts (Xing et al. 2004). HSV-1 amplicons also retain their ability for retrograde transport in neuronal axons allowing for the possibility of peripheral delivery for centrally located targets. For example inoculation of the foot pad in diabetic rats with HSV-1 amplicons expressing nerve growth factor (NGF) resulted in NGF expression in the dorsal root ganglion and protected against diabetes associated peripheral neuropathy (Goss et al. 2002).
One of the key concerns with any viral vector system is safety. HSV-1 amplicons are non-integrating viruses which thus avoids issues of cell transformation by insertional mutagenesis. The packaging of
Schematic showing packaging of OriS and pac containing plasmids into HSV-1 virions. A) AniBAC plasmid containing the gene of interst (GOI) is packaged using packaging virus to supply the HSV-1 genome in trans. This results in viral stocks that contain virions only carrying the iBAC GOI plasmid and wild-type-like virus. B) An improved packaging system using two plasmids in place of the wild-type-like virus. An oversized, ICP27 deleted BAC plasmid and a small plasmid that contains ICP27. This results in viral stocks that only contain virions with GOI containing iBAC.
HSV-1 delivers DNA to the cell as an extrachromosomal element and hybrid vectors have been designed to promote persistence of episomal vector DNA (Figure 2). The best described of these is the HSV-1/EBV hybrid vectors. The inclusion of the EBV latent origin of replication
The use of iBAC vectors in gene therapy is still evolving and a number of studies have demonstrated that these vectors are capable of efficient delivery and genetic complementation. Recent work has used the delivery and expression of the complete genomic of two genes key to the development of Alzheimer’s disease and Parkinson’s disease, microtubule associated protein tau (
Success had already been seen previously using iBAC vectors coding for the
Small cDNA-based vectors are not suitable to express loci which undergo complex splicing, such as the
HSV-1 amplicons have been used to deliver the complete genomic locus of the
The first example of
The
4. Physiologically-relevant gene expression vectors: use of native regulatory regions
Whole genomic loci represent an excellent means of ensuring physiologically-relevant expression in target cells. However, the large size of BAC plasmids precludes their use in all but a few viral vector systems. Although, non-viral systems such as hydrodynamic tail vein injection offer excellent means of delivery to target certain tissues, for many applications BAC-sized plasmids may not be practical. Many studies have attempted to combine the advantages of cDNA vectors (small size, high transduction or transfection efficiency, and high levels of protein expression) with an advantage of a whole genomic locus, being regulated physiologically-relevant expression. Depending on the gene of interest it may be necessary only to ensure expression of the transgene is restricted to a particular cell type; alternatively, it may be necessary to ensure transgene expression also tracks changes in cell physiology to ensure therapeutic and not pathologic transgene expression.
There has been extensive research into targeting gene expression to desired tissues using transcriptional restriction. Such work uses well-characterised promoters and enhancer regions that limit transgene expression to certain desired cell types where they are active. Liver-directed gene expression for example has been achieved using the promoter regions from either the albumin (Follenzi et al. 2002) or α1 antitrypsin genes (Le et al. 1997) to target expression of clotting factors to the liver to treat the haemophilia family of diseases. Targeting gene expression to cells in vascular wall is possible using endothelial cell restricted expression through the use of promoter such as VE-cadherin or VEGFR-1 (Quinn et al. 2000; Nicklin et al. 2001). Vascular smooth muscle specific expression has been achieved using promoters like the SM22 promoter (Imai et al. 2001) and was successfully used to target expression of heme oxygenase 1 to the vascular endothelium.
For some diseases it is not enough to limit expression to cell type. The temporal dynamics of gene expression is also important. One novel way of achieving physiological expression using small cDNA vectors is to generate a genomic mini-gene construct that uses native gene expression elements with a cDNA transgene. Wiskott-Aldrich syndrome (WAS) is an excellent example of the need for native regulatory elements to ensure correct expression dynamics. WAS is an X-linked recessive disease caused by mutations in the WAS protein gene (
One example in which clinical success has been seen with a vector containing native regulatory elements is in treatment of Leber’s congenital amaurosis, a group of recessive congenital rod-cone dystrophies. Mutations in a retinal pigment epithelium specific gene called
These two examples use only a minimal promoter region, which may be appropriate for those genes where regulatory elements are located in a small region proximal to the start of the coding region. However, as was seen in the WAS example, a larger portion of genomic DNA may be necessary for full physiological regulation. In our laboratory we have investigated the use of a 10 kb piece of genomic DNA to ensure fully physiological expression of the low density lipoprotein receptor gene (
FH represents a significant challenge for gene therapy due to the regulation of
We have previously shown that an iBAC vector containing the 135 kb
The challenge was to maintain physiologically-regulated expression while improving transfection efficiency using hydrodynamic tail vein injection. We built genomic DNA mini-gene vectors that contained 10 kb of genomic DNA encompassing the full genomic DNA promoter of the human
Liver-directed delivery of LDLR mini-gene vectors in vivo using hydrodynamic tail vein injection resulted in expression from the
This work describes the successful combination of genomic DNA regulatory elements with a mini-gene cDNA vector. Expression from this vector is physiologically-regulated by intracellular cholesterol levels. Delivery of the smaller-sized mini-gene vector is more efficient than with the full BAC and highlights the possibility of combining gene replacement gene therapy with traditional medical treatments. Combining gene delivery with treatment that will reduce the amount of cholesterol being synthesised by the liver could increase the power of the gene delivery ensuring efficient binding and internalisation of LDL to reduce plasma cholesterol levels. We have demonstrated
3. Conclusion
Gene replacement gene therapy has been under investigation for a number of years and is emerging as a potentially potent tool to treat genetic disease. Most gene therapy protocols involve the use of small cDNA vectors where expression of the transgene is constitutive and unregulated. While for some conditions this may be adequate, others will require the expression of therapeutic genes to be regulated spatially, temporally and physiologically to circumvent issues with genotoxicity, loss of expression, and lack of therapeutic effect in animal models.
Several advances have been made in recent years to address these issues. The use of transcriptional restriction is now wide-spread with many studies employing cell-specific promoters to ensure gene expression is limited to target cells. There have also been developments in the use of whole genomic DNA loci transgenes. This opens the possibility of using vectors for gene therapy which completely recapitulate endogenous expression. Advances in viral vectors based on helper virus-free HSV-1 amplicons mean that viral delivery of large genomic loci >100 kb is now possible in vivo. Finally, the development of novel vectors which incorporate genomic DNA elements to achieve physiological expression in a mini-gene vector format will push the use of genomic regulatory elements in gene therapy vectors closer to a clinical reality.
A greater body of work
Acknowledgments
Our work on gene therapy and vector development has been supported by the British Heart Foundation, the Medical Research Council, the Biotechnology and Biological Sciences Research Council, the Friedreich's Ataxia Research Alliance, and the Wellcome Trust.
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