original article
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Infectious Delivery and Expression of a 135 kb Human FRDA Genomic DNA Locus Complements Friedreich’s Ataxia Deficiency in Human Cells Silvia Gomez-Sebastian1, Alfredo Gimenez-Cassina2, Javier Diaz-Nido2, Filip Lim2 and Richard Wade-Martins1 1 The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK; 2Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma de Madrid, Madrid, Spain
Friedreich’s ataxia (FA) is the most common recessive ataxia, affecting 1–2 in 50,000 Caucasians, and there is currently no effective cure or treatment. FA results from a deficiency of the mitochondrial protein frataxin brought about by a repeat expansion in intron 1 of the FRDA gene. The main areas affected are the central nervous system (particularly the spinocerebellar system) and cardiac tissue. Therapies aimed at alleviating the neurological degeneration have proved unsuccessful to date. Here, we describe the construction and delivery of high capacity herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the entire 80 kb FRDA genomic locus, driven by the endogenous FRDA promoter and including all introns and flanking regulatory sequences within a 135 kb genomic DNA insert. FA patient primary fibroblasts deficient in frataxin protein and exhibiting sensitivity to oxidative stress were transduced at high efficiency by FRDA genomic locus vectors. Following vector transduction, expression of FRDA protein by immunofluorescence was shown. Finally, functional complementation studies demonstrated restoration of the wild-type cellular phenotype in response to oxidative stress in transduced FA patient cells. These results suggest the potential of the infectious bacterial artificial chromosome-FRDA vectors for gene therapy of FA. Received 10 July 2006; accepted 6 September 2006. doi:10.1038/sj.mt.6300021
INTRODUCTION Friedreich’s ataxia (FA) is the most common recessive ataxia in Caucasians,1 characterized by gait and limb ataxia, signs of upper motoneuron degeneration (arreflexia and dysarthria), and cardiac hypertrophy, subsequently leading to death in the fourth or fifth decade of life. The disease is caused by a deficiency in frataxin, a mitochondrial protein whose function has not been
well established, although it is known to participate in the biogenesis of the iron-sulfur clusters, important cofactors for a number of proteins of the mitochondrial electron transfer chain.2–4 Frataxin is also able to stimulate oxidative phosphorylation or iron storage.5 Therefore, frataxin is a key molecule affecting energy metabolism and mitochondrial function. The frataxin protein is encoded by the FRDA gene, also known as X25, which lies on chromosome 9q13 (OMIM: 606829), spanning about 80 kb of genomic DNA. FRDA consists of seven exons, namely the coding exons 1, 2, 3, 4, 5a, 5b, and non-coding exon 6, leading to the transcription of three different messenger RNAs. The size of the major transcript is 1.3 kb, which includes exons 1–5a. An abnormal overexpansion of variable length of a GAA triplet in intron 1 is responsible for the disease in over 95% of FA disease cases, as it leads to the generation of unusual three-stranded DNA structures (also known as ‘‘sticky DNA’’) that impair transcription, thus reducing frataxin levels below 30% of normal.6 A long-standing hypothesis is that FA is due to iron-mediated oxidative stress. Cultured fibroblasts from FA patients, as well as DYFH1-yeast, deleted for the FRDA homolog, exhibit an increased sensitivity to oxidative stress.7,8 Furthermore, in cells cultured from FA patients and then challenged with exogenous oxidative stress, superoxide dismutases induction was impaired, possibly making the cells even more prone to oxidative damage.8–10 This suggests that continuous oxidative stress damage owing to an impaired response to oxidative stress could further contribute to cell degeneration in the disease. Although some treatments based upon administration of antioxidants have had some success in treating the cardiac condition,11–13 therapies aimed at alleviating the neurological degeneration have failed so far.14 Therefore, the development of gene therapy strategies to treat the neurological disorder would be valuable. One recurrent problem with viral gene transfer is the lack of persistence of transgene expression. This is partially due to the use of viral promoters, which are known to result in strong, but transient gene expression, in many tissues in vivo. Thus, transgenes consisting of whole genomic loci hold great
Correspondence: Richard Wade-Martins, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. E-mail:
[email protected]
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promise for therapeutic use as their expression is regulated by native promoter, enhancer, and silencer elements, similar to that of the native chromosomal gene and this leads to persistent expression over long periods of time. The human genome is now covered by a near-complete physical map15 consisting of fully sequenced, overlapping bacterial artificial chromosome (BAC) and P1-based artificial chromosome clones, which can be analyzed through the public databases. However, delivery of genomic DNA inserts of 4100 kb in size is difficult especially to primary human cell cultures. Herpes simplex virus type 1 (HSV-1)-based amplicon vectors are replication-deficient plasmid vectors containing the replication origin (oris) and the packaging signal (pac) from HSV1.16–18 Amplicons have proven to be very efficient at transducing neuronal cells, both in vitro and in vivo.19,20 Additionally, as essentially ‘‘gutless’’ vectors carrying only B1% of the HSV-1 genome, HSV-1 amplicon vectors have a large transgene capacity to accommodate up to 150 kb of exogenous DNA. This unique property enables HSV-1 amplicon vectors to deliver whole genomic DNA loci for expression and analysis. The recently described infectious BAC (iBAC) system21 enables highly efficient HSV-1 amplicon-mediated delivery and expression of BAC library inserts in human and mouse cells.22–24 Here, we present a gene complementation strategy for FA based upon HSV-1-derived amplicon vectors carrying the complete FRDA locus (iBAC-FRDA). FA patient fibroblasts transduced with iBAC-FRDA vectors show high efficiency of delivery and expression of FRDA protein by immunofluorescence. Functional complementation studies show iBAC-FRDAmediated restoration of the wild-type phenotype in FA patient cells in response to oxidative stress. The results presented are encouraging and, in summary, show that infectious gene transfer of the complete FRDA locus results in physiological levels of gene expression and functional rescue in human cells.
RESULTS iBAC-FRDA vector construction A BAC clone (RP11-265B8) was identified which contains the entire 80 kb FRDA locus from human chromosome 9 within a 188 kb genomic insert. The HSV-1 amplicon has a maximum packaging capacity of approximately 155–160 kb, and so a simple restriction enzyme digest strategy was used to remove extreme 50 DNA sequence from the BAC insert. Two PmeI restriction enzyme sites within the genomic insert were identified (Figure 1a). PmeI restriction enzyme digestion and religation of RP11265B8 excised a 54 kb fragment of extreme 50 DNA sequence. The modified insert now contained approximately 38 kb of intact promoter region, the 80 kb FRDA locus, and 17 kb of downstream sequence. We then used a Cre/loxP-recombination strategy to incorporate the retrofitting vectors pHG and pEHHG into the FRDA BAC to produce two different iBAC-FRDA vectors. pHG contains the viral replication origin (oriS) and the packaging signal (pac) and a green fluorescent protein (GFP) cassette for monitoring delivery efficiency. In addition, pEHHG contains the episomal retention sequences from Epstein–Barr virus Molecular Therapy vol. 15 no. 2, feb. 2007
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Figure 1 Construction of iBAC-FRDA genomic DNA expression vectors. (a) Schematic representation of the structure of the BAC clone RP11-265B8 genomic DNA insert spanning 188 kb from human chromosome 9 containing the whole genomic FRDA locus encoding frataxin. The seven exons are marked as vertical lines. A 135 kb genomic DNA insert was obtained by PmeI digestion and religation. (b) The two retrofitting constructs used to convert the BAC into infectious vectors. pHG contains the HSV-1 amplicon elements oris and pac. In addition, pEHHG contains the episomal retention elements from Epstein–Barr virus, namely the Epstein–Barr virus latent origin of replication (oriP) and the Epstein–Barr virus nuclear antigen (EBNA-1) gene.
(Figure 1b). Correctly retrofitted clones were identified and named pHG-FRDA or pEHHG-FRDA. Both vectors were then packaged as infectious HSV-1 amplicon particles using an improved helper virus-free packaging system as described previously.25 Vector titers of B3 107 transducing units/ml were typically obtained for both iBAC vectors.
Functional delivery and expression of human frataxin from the iBAC-FRDA vector in mouse cells We first measured the efficiency of functional delivery of the human FRDA locus by the iBAC-FRDA vectors in neuronal-like mouse P19 cells which are widely used as a neuronal cell culture model in neurobiology-related studies. P19 cells were transduced by the iBAC-FRDA vectors (pHG-FRDA or pEHHG-FRDA) or the empty control vectors (pHG or pEHHG) at a multiplicity of infection (MOI) of 20. Vector transduction was assayed by fluorescence microscopy using the reporter GFP gene on the vector and shown to be approximately 40% (Figure 2a). Finally, reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to detect both human and murine frataxin using species-specific primer pairs (Figure 2b). Characterization of FRDA-deficient human fibroblasts Human FA patient cells typically have very low levels of FRDA messenger RNA and protein owing to decreased FRDA transcription.26 We characterized gene expression from two different FA patient primary fibroblast cell lines (GM03816 and 249
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GM04078) in comparison to the primary wild-type human fibroblast line MRC9. We demonstrated a deficiency in FRDA gene transcription by semiquantitative RT-PCR (Figure 3a) and protein expression by Western blot (Figure 3b) and immunofluorescence (Figure 3c) in GM03816 and GM04078. The two primary human FA patient fibroblasts GM03816, GM04078, and the wild-type fibroblast cell line MRC9, were tested for their ability to be transduced by the pEHHG and iBAC-FRDA vectors. We found that all cell lines were efficiently transduced by the amplicon vectors and that the cells maintain GFP expression for at least 1 week. Amplicon transduction was shown to be non-cytotoxic, with cells exhibiting 495% viability at MOIs of 10 and 30 as measured by staining of live and dead cells 1 week post-transduction (data not shown).
Restoration of frataxin protein expression in FA fibroblasts following iBAC-FRDA transduction FA is a disease caused by loss of function of the frataxin protein and so the iBAC system is suitable for restoring protein function at an appropriate level in frataxin-deficient cells. The presence of the whole genomic locus under the control of the native regulatory sequences, including promoter, enhancer, and silencer elements will likely produce physiological and stable expression of the deficient protein. The two primary FA human fibroblast cell lines were transduced by the pEHHG-FRDA iBAC vector at an MOI of 30 and the resulting frataxin expression was examined by immunofluoresence. We have observed that frataxin expression reached the highest levels between 4 and 7 days posttransduction (data not shown), which is consistent with reports 250
that the FRDA promoter is known to express at low levels under physiological regulation. Therefore, all expression and functional assays in human fibroblasts were conducted 1 week posttransduction. One week post-transduction with iBAC-FRDA FA cells showed restored expression of frataxin protein compared with control-untransduced cells (Figure 4). The frataxin expression level and pattern of subcellular localization observed in the iBAC-transduced FA fibroblasts were similar to that detected in the wild-type MRC9 fibroblasts.
Complementation by iBAC-FRDA delivery of susceptibility to oxidative stress in FA primary fibroblasts FA patient fibroblasts have been shown to exhibit biochemical deficiencies, including increased sensitivity to oxidative stress.8 Once we had confirmed restoration of frataxin protein expression in FA fibroblasts by iBAC-FRDA transduction, we next assayed the restoration of the wild-type oxidative stress response phenotype in transduced FA fibroblasts by two methods. First, it is known that cells affected by oxidative stress develop a disrupted plasma membrane which allows propidium iodide (PI) to stain the nucleus. This provides a simple assay of cell viability. FA fibroblasts were transduced by the control vector pEHHG, or by pEHHG-FRDA, at an MOI of 30, and susceptibility to oxidative stress following a 4-h exposure to 400 mM of H2O2 1 week post-transduction was measured (Figure 5). pEHHG-transduced cells express the GFP vector reporter gene (green) and also show PI nuclear staining (red). In contrast, pEHHG-FRDA-transduced FA fibroblasts withstand www.moleculartherapy.org vol. 15 no. 2, feb. 2007
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Figure 3 Characterization of FRDA transcription and frataxin expression in FA primary fibroblasts. (a) RT-PCR detection of human FRDA expression in wild-type primary human fibroblasts (MRC9), or in primary FA fibroblasts (GM03816, GM04078). FRDA transcript levels were quantified by measuring band intensities relative to GAPDH transcript levels in MRC9 and FA fibroblasts. (b) Expression analysis of frataxin protein and mitochondrial OxPhos Complex subunit a (OPC-V) of ATP synthase as an internal control by Western blot in wild-type and FA patient primary fibroblasts. (c) Expression analysis of frataxin protein (red) and OPC-V internal control (green) by immunocytochemistry in wild-type and FA patient primary fibroblasts.
the oxidative stress treatment without any sign of death, expressing GFP but not showing nuclear staining for PI (Figure 5). Second, FA fibroblasts transduced by either pEHHG or pEHHG-FRDA were exposed to a range of oxidative stress conditions and cell survival was measured by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (see Materials and Methods for details). FA fibroblasts were transduced by pEHHG or pEHHG-FRDA at an MOI of 60 and 7 days later were exposed to 200 or 400 mM of H2O2 for 6 h. Prior transduction with the pEHHG-FRDA genomic locus expression vector resulted in a highly significant increase in cell survival at both levels of H2O2 treatment (Figure 6a; Po0.01 by t-test). In a separate experiment, a very similar protective effect was observed following transduction of FA cells with either pEHHG or pEHHG-FRDA at an MOI of 90, followed 1 week later by exposure to 8 mM FeCl3 for 6 h (Figure 6b; Po0.01 by t-test). In Figure 6, the level of restoration of wild-type phenotype seen is in agreement with the level of B30% transduction reached in the transduction. This confirms a tight correlation between gene delivery efficiency and the level of phenotype correction and suggests that each genomic FRDA locus copy is delivered and expressed at a physiological relevant level. Taken together, our Molecular Therapy vol. 15 no. 2, feb. 2007
data clearly show that we are able to bring about a significant correction of the FA fibroblast phenotype after infectious delivery and expression of the whole FRDA locus using the iBAC-FRDA vector.
DISCUSSION The purpose of this study was to demonstrate the potential of a whole genomic locus delivery approach for functional complementation of the cellular and biochemical deficiency seen in FA cells. We have constructed iBAC-FRDA expression vectors carrying the whole genomic FRDA locus of 80 kb, flanked by 38 kb of 50 sequence and 17 kb of downstream. The vectors can be efficiently packaged to high titer (B3 107 transducing units/ml) by an improved helper virus-free packaging system for HSV-1 amplicons.25 A major advantage of the iBAC system lies in the ability to transfer efficiently by transduction genomic DNA inserts of 4100 kb to cells that are resistant to transfection methodologies.21 Here, we have demonstrated expression of human FRDA messenger RNA and protein following vector transduction in both mouse and human cells. We characterized in detail the phenotype of primary FA fibroblast cultures, and finally, we 251
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Figure 4 Restoration of frataxin protein expression in FA patient primary fibroblasts following iBAC-FRDA transduction. Immunocytochemistry analysis of FA fibroblasts GM03816 and GM04078 transduced by pEHHG-FRDA at an MOI of 30 shows restoration of human frataxin expression (red) and OPC-V internal control (green) 1 week post-transduction. Arrows indicate FA patient cells staining positive for FRDA protein.
showed significant restoration of the wild-type oxidative stress resistance phenotype in primary FA fibroblasts otherwise extremely sensitive to such conditions. The percentage of restoration agrees closely with the efficiency of the transduction reached in the assay (B30%) confirming a tight correlation between gene delivery efficiency and the level of phenotype correction. This work is in close agreement with our previous studies in which we demonstrate physiological levels of gene expression from a complete genomic locus within a gene expression vector.21–23,27 This is expected when expression from the transgene is driven by the endogenous promoter, surrounded by native enhancer and silencer elements and including all the introns and exons in the correct genomic DNA context. The level of frataxin protein expression and subcellular localization observed in the transduced FA fibroblasts was similar to that detected in the wild-type fibroblasts indicating, as expected, that we had obtained physiologically relevant levels of transgene expression. Obtaining a physiological level of expression of FRDA is important, as some authors have suggested that overexpression of the FRDA protein is toxic.28 In the previous study, FRDA cDNA expression cassettes were introduced into FA patient fibroblasts using lentiviral or adeno-associated viral vectors. Very high levels of FRDA expression were observed which were thought to be toxic to transduced cells.28 Taken together, our data clearly show that we are able to effect a significant rescue of the FA fibroblast phenotype after transduction with the whole FRDA locus. 252
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Figure 5 iBAC-FRDA transduction restores the resistance of individual FA primary fibroblasts to oxidative stress. FA human fibroblasts GM03816 and GM04078 transduced by pEHHG-FRDA, but not pEHHG, show enhanced viability measured by PI staining after exposure to 4 h of treatment with H2O2 400 mM. pEHHG-FRDA-transduced cells (GFPpositive green cells) maintain cell membrane integrity and thus do not permit entry of PI to produce red nuclear staining. Vector transductions were performed at an MOI of 30.
Within the 135 kb iBAC-FRDA genomic DNA insert we have used, there is one other complete, very small, gene in the insert called PRKACG which lies 22 kb upstream of FRDA (see http://genome.ucsc.edu/). PRKACG is a single exon gene of 1,558 bp, which encodes for the cyclic adenosine monophosphate-dependent protein kinase (PKA) catalytic gamma subunit. It is highly unlikely that expression of PRKACG will affect FRDA function in cells. It is known that mutations within the FRDA gene reduce FRDA expression to cause FA and give rise to the FA biochemical defect. In our study, we have shown that FA patient cells do not express FRDA protein before transduction, and do afterwards. No mechanism is known to link PRKACG to FRDA function and we conclude that the likelihood of PRKACG expression from our genomic DNA insert affecting the FA cellular phenotype is negligible. The last two exons from the 30 end of another gene, PIP5K1B, a large 300 kb, 16 exon gene lie upstream of FRDA within the iBACFRDA insert. These last two exons in isolation have no mechanism of expression and hence will not interfere with our experiment. Treatment options for FA as well as other inherited and acquired conditions involving pathology in post-nuclear supernatant sensory neurons remain inadequate. In the case of FA, some treatments based upon administration of antioxidants have had modest success at treating the cardiac condition.11–13 However, therapies aimed at alleviating the neurological degeneration have failed so far.14 Therefore, gene therapy delivered by a neurotropic vector, such as HSV-1, is a promising www.moleculartherapy.org vol. 15 no. 2, feb. 2007
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Vector construction. The fully sequenced BAC RP11-265B8 clone (Gene Bank Accession number: AL162730) was obtained from the BACPAC Resources Center (http://bacpac.chori.org/). Cre/loxPmediated retrofitting of pHG and pEHHG was performed as described previously.21,25 Tissue culture. FA fibroblasts (GM03816 and GM04078) were obtained
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