Publications by authors named "Samantha L Ginn"

24 Publications

  • Page 1 of 1

Genome editing in the human liver: Progress and translational considerations.

Prog Mol Biol Transl Sci 2021 6;182:257-288. Epub 2021 Mar 6.

Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Westmead, NSW, Australia; Discipline of Child and Adolescent Health, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia. Electronic address:

Liver-targeted genome editing offers the prospect of life-long therapeutic benefit following a single treatment and is set to rapidly supplant conventional gene addition approaches. Combining progress in liver-targeted gene delivery with genome editing technology, makes this not only feasible but realistically achievable in the near term. However, important challenges remain to be addressed. These include achieving therapeutic levels of editing, particularly in vivo, avoidance of off-target effects on the genome and the potential impact of pre-existing immunity to bacteria-derived nucleases, when used to improve editing rates. In this chapter, we outline the unique features of the liver that make it an attractive target for genome editing, the impact of liver biology on therapeutic efficacy, and disease specific challenges, including whether the approach targets a cell autonomous or non-cell autonomous disease. We also discuss strategies that have been used successfully to achieve genome editing outcomes in the liver and address translational considerations as genome editing technology moves into the clinic.
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http://dx.doi.org/10.1016/bs.pmbts.2021.01.030DOI Listing
March 2021

Restoring the natural tropism of AAV2 vectors for human liver.

Sci Transl Med 2020 09;12(560)

Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte-derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.
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http://dx.doi.org/10.1126/scitranslmed.aba3312DOI Listing
September 2020

Efficient editing of OTC-deficient patient-derived primary human hepatocytes.

JHEP Rep 2020 Feb 27;2(1):100065. Epub 2019 Dec 27.

Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Westmead, Australia.

Background & Aims: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase () locus in patient-derived primary human hepatocytes at therapeutically relevant levels.

Methods: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human locus were first evaluated against an minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native locus in patient-derived primary human hepatocytes xenografted into the FRG ( ) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence.

Results: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes , locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 -predicted sites in the genome.

Conclusions: We report efficient single-nucleotide correction of a disease-causing mutation in the locus in patient-derived primary human hepatocytes at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events.

Lay Summary: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient correction of a patient-specific disease-causing mutation in the gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice.
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http://dx.doi.org/10.1016/j.jhepr.2019.100065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005564PMC
February 2020

Bone Marrow Transplantation for Treatment of the Col1a2 Osteogenesis Imperfecta Mouse Model.

Calcif Tissue Int 2019 04 8;104(4):426-436. Epub 2018 Dec 8.

Orthopaedic Research and Biotechnology Unit, Kids Research, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.

Bone marrow transplantation (BMT) of healthy donor cells has been postulated as a strategy for treating osteogenesis imperfecta (OI) and other bone fragility disorders. The effect of engraftment by tail vein injection and/or marrow ablation by 6 Gy whole body irradiation were tested in Col1a2 (OI) mice as a model of mild-moderate OI. Dual-emission X-ray absorptiometry, microCT, and 4-point bending were used to measure bone volume (BV), bone mineral density (BMD), and biomechanical strength. BV, BMD, and mechanical strength were reduced in OI mice compared to wild type (WT) controls. BMT with and without irradiation yielded no difference in BV and BMD outcomes for both OI and WT mice, at 3 weeks. Transplantation of OI cells into OI mice to test for paracrine effects of BMT also showed no difference with non-transplanted OI mice. In a parallel cell tracking study, donor marrow was taken from transgenic mice constitutively expressing tdTomato and transplanted into WT mice. Lineage tracking demonstrated that irradiation considerably enhanced engraftment of tdTomato+ cells. However, tdTomato+ cells predominantly expressed TRAP and not AP, indicating engrafted donor cells were chiefly from the hematopoietic lineages. These data show that whole marrow transplantation fails to rescue the bone phenotype of Col1a2 (OI) mice and that osteopoietic engraftment is not significantly enhanced by irradiation. These findings are highly relevant to modern approaches focused on the gene repair of patient cells ex vivo and their subsequent reintroduction into the osteopoietic compartment via the circulation.
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http://dx.doi.org/10.1007/s00223-018-0504-3DOI Listing
April 2019

Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality.

Mol Ther Methods Clin Dev 2019 Mar 1;12:71-84. Epub 2018 Nov 1.

Translational Vectorology Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.

Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications.
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http://dx.doi.org/10.1016/j.omtm.2018.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279885PMC
March 2019

Thymocyte self-renewal and oncogenic risk in immunodeficient mouse models: relevance for human gene therapy clinical trials targeting haematopoietic stem cell populations?

Mamm Genome 2018 12 4;29(11-12):771-776. Epub 2018 Sep 4.

Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Locked Bag 2023, Wentworthville, NSW, 2145, Australia.

Emerging evidence indicates that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Here we discuss formative studies demonstrating that, in mice, early thymocytes acquire self-renewing potential when thymic progenitor supply is sub-physiological and the importance of cellular competition with this at-risk cell population to prevent lymphoid malignancy. We also consider the possibility that increased thymic residency time, established under conditions of limited cellular competition, may have contributed to oncogenesis observed in early SCID-X1 trials when combined with insertional activation of proto-oncogenes such as LMO2.
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http://dx.doi.org/10.1007/s00335-018-9780-5DOI Listing
December 2018

Gene therapy clinical trials worldwide to 2017: An update.

J Gene Med 2018 05 19;20(5):e3015. Epub 2018 Apr 19.

Department of Laboratory Medicine, Uppsala University Hospital, Uppsala, Sweden.

To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our November 2017 update, we have entries on 2597 trials undertaken in 38 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and the genes that have been transferred. Details of the analyses presented, and our searchable database are available via The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in gene therapy clinical trials around the world, and discuss key trends since the previous review, namely the use of chimeric antigen receptor T cells for the treatment of cancer and advancements in genome editing technologies, which have the potential to transform the field moving forward.
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http://dx.doi.org/10.1002/jgm.3015DOI Listing
May 2018

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Nat Genet 2017 Aug 19;49(8):1267-1273. Epub 2017 Jun 19.

Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, University of Sydney, Sydney, New South Wales, Australia.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
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http://dx.doi.org/10.1038/ng.3893DOI Listing
August 2017

Limiting Thymic Precursor Supply Increases the Risk of Lymphoid Malignancy in Murine X-Linked Severe Combined Immunodeficiency.

Mol Ther Nucleic Acids 2017 Mar 10;6:1-14. Epub 2016 Dec 10.

Gene Therapy Research Unit, Children's Medical Research Institute, The University of Sydney and The Sydney Children's Hospitals Network, Westmead, NSW 2145, Australia; Discipline of Child and Adolescent Health, The University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

In early gene therapy trials for SCID-X1, using γ-retroviral vectors, T cell leukemias developed in a subset of patients secondary to insertional proto-oncogene activation. In contrast, we have reported development of T cell leukemias in SCID-X1 mice following lentivirus-mediated gene therapy independent of insertional mutagenesis. A distinguishing feature in our study was that only a proportion of transplanted γc-deficient progenitors were transduced and therefore competent for reconstitution. We hypothesized that reconstitution of SCID-X1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy. To test this hypothesis, in the absence of transduction, SCID-X1 mice were reconstituted with serially fewer wild-type hematopoietic progenitors. A robust inverse correlation between hematopoietic progenitor cell dose and T-lymphoid malignancy was observed, with earlier disease onset at lower cell doses. Malignancies were of donor origin and carried activating Notch1 mutations. These findings align with emerging evidence that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Although insertional proto-oncogene activation is required for the development of malignancy in humans, failure of γc-deficient thymocytes to effectively compete with this at-risk cell population may have also contributed to oncogenesis observed in early SCID-X1 trials.
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http://dx.doi.org/10.1016/j.omtn.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363493PMC
March 2017

Coherence analysis discriminates between retroviral integration patterns in CD34(+) cells transduced under differing clinical trial conditions.

Mol Ther Methods Clin Dev 2015 29;2:15015. Epub 2015 Apr 29.

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead , Westmead, Australia ; The University of Sydney, Discipline of Paediatrics and Child Health , Westmead, Australia.

Unequivocal demonstration of the therapeutic utility of γ-retroviral vectors for gene therapy applications targeting the hematopoietic system was accompanied by instances of insertional mutagenesis. These events stimulated the ongoing development of putatively safer integrating vector systems and analysis methods to characterize and compare integration site (IS) biosafety profiles. Continuing advances in next-generation sequencing technologies are driving the generation of ever-more complex IS datasets. Available bioinformatic tools to compare such datasets focus on the association of integration sites (ISs) with selected genomic and epigenetic features, and the choice of these features determines the ability to discriminate between datasets. We describe the scalable application of point-process coherence analysis (CA) to compare patterns produced by vector ISs across genomic intervals, uncoupled from association with genomic features. To explore the utility of CA in the context of an unresolved question, we asked whether the differing transduction conditions used in the initial Paris and London SCID-X1 gene therapy trials result in divergent genome-wide integration profiles. We tested a transduction carried out under each condition, and showed that CA could indeed resolve differences in IS distributions. Existence of these differences was confirmed by the application of established methods to compare integration datasets.
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http://dx.doi.org/10.1038/mtm.2015.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445430PMC
June 2015

Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence.

Nucleic Acids Res 2014 10;42(16):e129. Epub 2014 Jul 10.

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.

Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved.
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http://dx.doi.org/10.1093/nar/gku607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176369PMC
January 2015

Gene therapy clinical trials worldwide to 2012 - an update.

J Gene Med 2013 Feb;15(2):65-77

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, NSW, Australia.

To date, over 1800 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical trials from official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our June 2012 update, we have entries on 1843 trials undertaken in 31 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are available on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in clinical trials of gene therapy approaches around the world and discuss the prospects for the future.
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http://dx.doi.org/10.1002/jgm.2698DOI Listing
February 2013

Gene therapy: progress in childhood disease.

J Paediatr Child Health 2012 Jun 21;48(6):466-71. Epub 2011 Oct 21.

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Australia.

The recent sequencing of the human genome combined with the development of massively high throughput genetic analysis technologies is driving unprecedented growth in our knowledge of the molecular basis of disease. While this has already had a major impact on our diagnostic power, the therapeutic benefits remain largely unrealised. This review examines progress in the exciting and challenging field of gene therapy. In particular we focus on the treatment of genetic disease in infants and children where the most significant successes have been observed to date, despite the majority of trial participants being adults. Notably, gene transfer to the haematopoietic compartment has provided the clearest examples of therapeutic benefit, particularly in the context of primary immunodeficiencies. The triumphs and tribulations of these successes are explored, and the key challenges confronting researchers as they seek to further advance the field are defined and discussed.
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http://dx.doi.org/10.1111/j.1440-1754.2011.02204.xDOI Listing
June 2012

Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and gammac overexpression.

Mol Ther 2010 May 30;18(5):965-76. Epub 2010 Mar 30.

Gene Therapy Research Unit of the Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.

The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in gammac-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing gammac from the phosphoglycerate kinase (PGK) and Wiscott-Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-alpha (EF1alpha) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or gammac overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.
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http://dx.doi.org/10.1038/mt.2010.50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890120PMC
May 2010

Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a.

BMC Musculoskelet Disord 2009 May 15;10:51. Epub 2009 May 15.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Sydney, NSW, Australia.

Background: Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9), but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression.

Methods: We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD) were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes (alkaline phosphatase, osteocalcin and bone morphogenetic protein receptor-1A) as measured by quantitative PCR.

Results: BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated Bmpr-1a expression. Chronic BMP-2 treatment resulted in peak Bmpr-1a expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of Bmpr-1a that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine BMPR-1A/Bmpr-1a. Forced myogenic gene expression in fibroblasts was associated with a significant increase in Bmpr-1a expression and a synergistic increase in the osteogenic response to BMP-2.

Conclusion: These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the variable response of different cell lineages to BMP-2.
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http://dx.doi.org/10.1186/1471-2474-10-51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685768PMC
May 2009

Gene transfer of connexin43 mutants attenuates coupling in cardiomyocytes: novel basis for modulation of cardiac conduction by gene therapy.

Circ Res 2007 Jun 10;100(11):1597-604. Epub 2007 May 10.

Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, Australia.

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.
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http://dx.doi.org/10.1161/CIRCRESAHA.106.144956DOI Listing
June 2007

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb.

J Gene Med 2007 Jun;9(6):429-39

Department of Respiratory Medicine, The Children's Hospital at Westmead, Westmead, Sydney, NSW, Australia.

Background: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges.

Methods: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway.

Results: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low.

Conclusion: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.
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http://dx.doi.org/10.1002/jgm.1039DOI Listing
June 2007

Limiting {gamma}c expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors.

Blood 2007 Jul 15;110(1):91-8. Epub 2007 Mar 15.

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Australia.

X-linked severe combined immunodeficiency (SCID-X1) results from mutations in the IL2RG gene, which encodes the common gamma chain (gammac) of the receptors for interleukin (IL)-2, 4, 7, 9, 15, and 21. Affected infants typically lack T and natural killer (NK) cells as a consequence of loss of signaling via the IL-7 receptor (IL-7R) and the IL-15R, respectively. In some infants, however, autologous NK cells are observed despite failure of T-cell ontogeny. The mechanisms by which mutations in gammac differentially impact T- and NK-cell ontogeny remain incompletely understood. We used SCID-X1 patient-derived EBV-transformed B cells to test the hypothesis that the IL-15R-mediated signaling is preferentially retained as gammac expression becomes limiting. Signal transduction via the IL-15R was readily detected in control EBV-transformed B cells, and via the IL-7R when modified to express IL-7Ralpha. Under the same experimental conditions, patient-derived EBV-transformed B cells expressing trace amounts of gammac proved incapable of signal transduction via the IL-7R while retaining the capacity for signal transduction via the IL-15R. An equivalent result was obtained in ED-7R cells modified to express varying levels of gammac. Collectively, these results confirm that signal transduction via the IL-15R, and hence NK ontogeny, is preferentially retained relative to the IL-7R as gammac expression becomes limiting.
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http://dx.doi.org/10.1182/blood-2006-11-055442DOI Listing
July 2007

Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

Hum Gene Ther 2005 Aug;16(8):947-56

Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Sydney, NSW, Australia.

Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.
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http://dx.doi.org/10.1089/hum.2005.16.947DOI Listing
August 2005

Treatment of an infant with X-linked severe combined immunodeficiency (SCID-X1) by gene therapy in Australia.

Med J Aust 2005 May;182(9):458-63

Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Unit, Sydney, NSW.

Objective: To report the outcome of gene therapy in an infant with X-linked severe combined immunodeficiency (SCID-X1), which typically causes a lack of T and natural killer (NK) cells.

Design And Setting: Ex-vivo culture and gene transfer procedures were performed at The Children's Hospital at Westmead, Sydney, NSW, in March 2002. Follow-up to March 2005 (36 months) is available.

Patient: A 9-month-old male infant with confirmed SCID-X1 (including complete absence of T cells) with an NK+ phenotype (a less common variant of SCID-X1), and no HLA-identical sibling donor available for conventional bone marrow transplantation.

Procedure: CD34+ haemopoietic progenitor cells were isolated from harvested bone marrow and cultured with cytokines to stimulate cellular replication. Cells were then genetically modified by exposure to a retrovirus vector encoding human gamma c (the common gamma chain of several interleukin receptors; mutations affecting the gamma c gene cause SCID-X1). Gene-modified cells (equivalent to 1.3 x 10(6) CD34+/gamma c+ cells/kg) were returned to the infant via a central line.

Results: T cells were observed in peripheral blood 75 days after treatment, and levels increased rapidly to 0.46 x 10(9) CD3+ cells/L at 5 months. Within 2 weeks of the appearance of T cells, there was a distinct clinical improvement, with early weight gain and clearance of rotavirus from the gut. However, T-cell levels did not reach the reference range, and immune reconstitution remained incomplete. The infant failed to thrive and developed weakness, hypertonia and hyperreflexia in the legs, possibly the result of immune dysregulation. He went on to receive a bone marrow transplant from a matched unrelated donor 26 months after gene therapy.

Conclusions: This is the first occasion that gene therapy has been used to treat a genetic disease in Australia. Only partial immunological reconstitution was achieved, most likely because of the relatively low dose of gene-corrected CD34+ cells re-infused, although viral infection during the early phase of T-cell reconstitution and the infant's NK+ phenotype may also have exerted an effect.
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http://dx.doi.org/10.5694/j.1326-5377.2005.tb06785.xDOI Listing
May 2005

Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.

Circulation 2005 Feb;111(4):394-8

Department of Cardiology, Westmead Hospital, Westmead, Australia.

Background: Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling.

Methods And Results: High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication.

Conclusions: Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.
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http://dx.doi.org/10.1161/01.CIR.0000153812.64956.EFDOI Listing
February 2005

A novel splice-site mutation in the common gamma chain (gammac) gene IL2RG results in X-linked severe combined immunodeficiency with an atypical NK+ phenotype.

Hum Mutat 2004 May;23(5):522-3

Gene Therapy Research Unit of the Children's Medical Research Institute and The Children's Hospital at Westmead, NSW, Australia.

Mutations in the gene encoding the common gamma chain (gammac) of interleukin receptors 2, 4, 7, 9, 15 and 21 result in X-linked severe combined immunodeficiency (SCID-X1). Classically, this disease is characterised by an absence of T and NK cells, and near normal numbers of functionally deficient B cells (B(+), T(-), NK(-) phenotype). Atypical phenotypes have also been described, but relatively little is known about the mechanisms by which the underlying mutations impair gammac-dependent interleukin receptor signalling to produce these disease variants. Here we describe a novel splice-site mutation resulting in the presence of near normal numbers of functionally deficient NK cells (B(+), T(-), NK(+) phenotype), in a SCID-X1 infant who was subsequently treated by gene therapy. The mutation, c.468+3A>C affecting the third base of intron 3 in the IL2RG gene, was shown to result in the production of two aberrantly spliced gammac mRNA species and reduction of correctly spliced message to trace levels, consistent with failure to detect gammac on the surface of B and NK cells by FACS analysis. The infant's genotype-phenotype correlation supports the hypothesis that interleukin 15 receptor-mediated signalling is preferentially retained as the amount of cell surface gammac becomes limiting. The possible implications for immunological reconstitution following gene therapy are also discussed.
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http://dx.doi.org/10.1002/humu.9235DOI Listing
May 2004

Promoter interference mediated by the U3 region in early-generation HIV-1-derived lentivirus vectors can influence detection of transgene expression in a cell-type and species-specific manner.

Hum Gene Ther 2003 Aug;14(12):1127-37

Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, NSW, Australia 2145.

In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency virus type 1 (HIV-1) accessory proteins. Cell-type specificity of CMV promoter expression, tropism of the VSV-G envelope, and blocks to molecular transduction were also precluded as possible mechanisms, thereby implicating transcriptional repression of the internal heterologous promoter. This promoter interference effect was found to be mediated by cis-acting sequences upstream of the core promoter elements located in the U3 region of the proviral long terminal repeats (LTRs). Deletion of this region, as in late-generation self-inactivating (SIN) lentivirus vectors, relieves this effect. This provides a basis for reevaluating data produced using early-generation U3-bearing lentivirus vectors and for reconciling these with results obtained using more contemporary SIN lentivirus vectors carrying a U3 deletion.
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http://dx.doi.org/10.1089/104303403322167975DOI Listing
August 2003

Dorsal root ganglia sensory neurons.

Methods Mol Biol 2003 ;229:155-67

Gene Therapy Unit of The Children's Hospital at Westmead and Children's Medical Research Institute, Sydney, Australia.

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http://dx.doi.org/10.1385/1-59259-393-3:155DOI Listing
October 2003
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