ORCID Profile
0000-0002-4772-8651
Current Organisations
Children's Medical Research Institute
,
Military Institute of Medicine
,
University of Sydney
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Related Links
Publications
The self-peptide repertoire plays a critical role in transplant tolerance induction
Publisher: Cold Spring Harbor Laboratory
Date: 09-11-2020
DOI: 10.1101/2020.11.09.359968
Abstract: While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8 + T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2K b -associated peptides recognised by CD8 + T cells from B10.BR (H-2 k ) mice, 13 of which were also recognised by BALB/c (H-2 d ) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.
Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-12-2012
DOI: 10.1161/CIRCRESAHA.112.274969
Abstract: Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression. To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging. We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine. Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.
Supplying Clotting Factors From Hematopoietic Stem Cell–derived Erythroid and Megakaryocytic Lineage Cells
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1038/MT.2009.238
Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution
Publisher: Elsevier BV
Date: 03-2023
Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton
Publisher: Elsevier BV
Date: 12-2019
In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation
Publisher: Elsevier BV
Date: 07-2018
Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in β-thalassemic mice
Publisher: American Society of Hematology
Date: 15-12-2007
DOI: 10.1182/BLOOD-2007-08-108647
Abstract: Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with β-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in β-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.
Globin gene transfer: a paradigm for transgene regulation and vector safety
Publisher: Brill
Date: 08-2003
Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing
Publisher: Springer Science and Business Media LLC
Date: 25-09-2020
DOI: 10.1038/S41467-020-18715-Y
Abstract: Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.
Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors
Publisher: Mary Ann Liebert Inc
Date: 04-2023
DOI: 10.1089/HUM.2022.188
Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8
Publisher: Elsevier BV
Date: 03-2022
Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation
Publisher: Wiley
Date: 14-12-2020
DOI: 10.1111/XEN.12669
Abstract: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non‐human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof‐of‐concept experiments the potential of the cytoplasmic ubiquitin‐editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF‐stimulated NF‐κB activation and NPI graft function. As adeno‐associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. Forcing the expression of A20 in NPI with Ad5 vector blocked NF‐κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro‐inflammatory genes Cxcl10 and Icam1 . A20‐expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14‐day culture period maximized NPI transduction efficiency ( % transduction rate), and suppressed NF‐κB‐dependent gene expression without adverse impact upon NPI maturation. We report a new protocol that allows for high‐efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.
Current status of globin gene therapy for the treatment of β‐thalassaemia
Publisher: Wiley
Date: 09-04-2008
DOI: 10.1111/J.1365-2141.2008.07098.X
Abstract: beta-Thalassaemia major is a congenital anaemia for which there is presently no curative therapy other than allogeneic haematopoietic stem cell transplantation. This therapeutic option, however, is not available to most subjects for whom there is no available human leucocyte antigen-matched bone marrow donor. The transfer of a regulated globin gene in autologous haematopoietic stem cells is therefore a directly needed alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation- and stage-restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, it has been demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression in vivo, resulting in the correction of anaemia and secondary organ damage in beta-thalassaemic mice. Several groups have extended these findings to various models of beta-thalassaemia and sickle cell disease. Different globin vectors, however, do not express beta-globin at the same level, and accordingly require the delivery of markedly different vector copy numbers to correct anaemia. Insulators are under investigation to assess whether they might enhance globin gene expression or vector safety. Altogether, recent advances in globin vector design bode well for upcoming clinical trials to assess the therapeutic value of globin gene transfer.
Directed Evolution of Adeno-Associated Virus Vectors in Human Cardiomyocytes for Cardiac Gene Therapy
Publisher: Elsevier BV
Date: 11-2018
Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome
Publisher: Springer Science and Business Media LLC
Date: 19-06-2017
DOI: 10.1038/NG.3893
Abstract: 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.
Somatic Correction of Junctional Epidermolysis Bullosa by a Highly Recombinogenic AAV Variant
Publisher: Elsevier BV
Date: 04-2014
DOI: 10.1038/MT.2013.290
Liver splitting during normothermic machine perfusion: a novel method to combine the advantages of both in-situ and ex-vivo techniques
Publisher: Elsevier BV
Date: 05-2023
TransientIn Vivoβ-Globin Production After Lentiviral Gene Transfer to Hematopoietic Stem Cells in the Nonhuman Primate
Publisher: Mary Ann Liebert Inc
Date: 06-2009
DOI: 10.1089/HUM.2008.186
The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis
Publisher: Elsevier BV
Date: 03-2021
DOI: 10.1053/J.GASTRO.2020.11.011
Abstract: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been h ered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5 Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.
rAAV-Mediated Tumorigenesis: Still Unresolved After an AAV Assault
Publisher: Elsevier BV
Date: 11-2012
DOI: 10.1038/MT.2012.220
The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA
Publisher: Oxford University Press (OUP)
Date: 08-02-2013
DOI: 10.1093/NAR/GKT068
Long-term ex-vivo normothermic perfusion of human split livers: a unique model to study new therapeutics and increase the number of available organs
Publisher: Research Square Platform LLC
Date: 02-03-2023
DOI: 10.21203/RS.3.RS-2610298/V1
Abstract: Current perfusion technology only allows livers to be preserved ex-vivo for short periods. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed develop a long-term model of ex-vivo perfusion including a surgical split and simultaneous perfusion of both partial grafts. Our long-term perfusion system included long-term oxygenators, a gas-mixer and a dialysis filter. Human livers declined for transplantation were perfused using a red-cell based perfusate under normothermic conditions (36°C) and then split and simultaneously perfused on separate machines. Ten human livers were split resulting in 20 partial grafts. The median ex-vivo survival was 165 hours (7 days). Long-term graft survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. The grafts that survived 7 days demonstrated significantly higher bile production, Factor-V production, and hepatic arterial flow and significantly lower microvesicular steatosis. We report reliable long-term ex-vivo perfusion of human livers and demonstrate the ability to split and perfuse these organs using a reproducible protocol. This provides the opportunity for improved assessment of organs and could act as a model for the testing of therapeutics with a matched control.
Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes
Publisher: Elsevier BV
Date: 02-2020
A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference
Publisher: Springer Science and Business Media LLC
Date: 25-12-2005
DOI: 10.1038/NBT1176
Abstract: The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.
A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus
Publisher: Mary Ann Liebert Inc
Date: 12-2019
Abstract: Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only
Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Recapitulates Long QT Phenotype for Drug Testing
Publisher: Elsevier BV
Date: 08-2014
Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle
Publisher: Elsevier BV
Date: 09-2018
Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids.
Publisher: Elsevier BV
Date: 06-2021
High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction
Publisher: Mary Ann Liebert Inc
Date: 05-2020
DOI: 10.1089/HUM.2019.264
Whole-genome landscapes of major melanoma subtypes
Publisher: Springer Science and Business Media LLC
Date: 05-2017
DOI: 10.1038/NATURE22071
Abstract: Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals erse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
AAV Vectors Containing rDNA Homology Display Increased Chromosomal Integration and Transgene Persistence
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1038/MT.2012.157
AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression
Publisher: Mary Ann Liebert Inc
Date: 06-2022
DOI: 10.1089/HUM.2021.278
Abstract: The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an
Progress Toward the Genetic Treatment of the β‐Thalassemias
Publisher: Wiley
Date: 11-2005
Abstract: The beta-thalassemias are congenital anemias that are caused by mutations that reduce or abolish expression of the beta-globin gene. They can be cured by allogeneic hematopoietic stem cell (HSC) transplantation, but this therapeutic option is not available to most patients. The transfer of a regulated beta-globin gene in autologous HSCs is a highly attractive alternative treatment. This strategy, which is simple in principle, raises major challenges in terms of controlling expression of the globin transgene, which ideally should be erythroid specific, differentiation- and stage-restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, May et al. demonstrated in 2000 that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have by now replicated and extended these findings to various mouse models of severe hemoglobinopathies, thus fueling enthusiasm for a potential treatment of beta-thalassemia based on globin gene transfer. Current investigation focuses on safety issues and the need for improved vector production methodologies. The safe implementation of stem cell-based gene therapy requires the prevention of the formation of replication-competent viral genomes and minimization of the risk of insertional oncogenesis. Importantly, globin vectors, in which transcriptional activity is highly restricted, have a lesser risk of activating oncogenes in hematopoietic progenitors than non-tissue-specific vectors, by virtue of their late-stage erythroid specificity. As such, they provide a general paradigm for improving vector safety in stem cell-based gene therapy.
RNA interference–induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice
Publisher: Springer Science and Business Media LLC
Date: 02-04-2016
DOI: 10.1038/NM.4079
Human COL7A1 -corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa
Publisher: American Association for the Advancement of Science (AAAS)
Date: 26-11-2014
DOI: 10.1126/SCITRANSLMED.3009540
Abstract: Patient-specific iPSCs were derived, corrected, and differentiated into human skin grafts for the treatment of recessive dystrophic epidermolysis bullosa.
An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.
Publisher: Elsevier BV
Date: 10-2018
Long-term ex situ normothermic perfusion of human split livers for more than 1 week
Publisher: Springer Science and Business Media LLC
Date: 08-08-2023
DOI: 10.1038/S41467-023-40154-8
Abstract: Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.
Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation
Publisher: Research Square Platform LLC
Date: 24-07-2023
DOI: 10.21203/RS.3.RS-3158964/V1
Abstract: Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.
A human adenovirus encoding IFN-γ can transduce Tasmanian devil facial tumour cells and upregulate MHC-I
Publisher: Microbiology Society
Date: 11-2022
DOI: 10.1099/JGV.0.001812
Abstract: The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil ( Sarcophilus harrisii ) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100 %. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-γ) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-γ was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2 and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as a vaccine platform for devils and potentially other marsupials.
Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality
Publisher: Elsevier BV
Date: 03-2019
Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity
Publisher: Springer Science and Business Media LLC
Date: 12-12-2017
DOI: 10.1038/S41467-017-01519-Y
Abstract: Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising ersity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
The self-peptide repertoire plays a critical role in transplant tolerance induction
Publisher: American Society for Clinical Investigation
Date: 11-2021
DOI: 10.1172/JCI146771
Neurological Disorders Associated with WWOX Germline Mutations—A Comprehensive Overview
Publisher: MDPI AG
Date: 07-04-2021
Abstract: The transcriptional regulator WW domain-containing oxidoreductase (WWOX) is a key player in a number of cellular and biological processes including tumor suppression. Recent evidence has emerged associating WWOX with non-cancer disorders. Patients harboring pathogenic germline bi-allelic WWOX variants have been described with the rare devastating neurological syndromes autosomal recessive spinocerebellar ataxia 12 (SCAR12) (6 patients) and WWOX-related epileptic encephalopathy (DEE28 or WOREE syndrome) (56 patients). In iduals with these syndromes present with a highly heterogenous clinical spectrum, the most common clinical symptoms being severe epileptic encephalopathy and profound global developmental delay. Knowledge of the underlying pathophysiology of these syndromes, the range of variants of the WWOX gene and its genotype-phenotype correlations is limited, h ering therapeutic efforts. Therefore, there is a critical need to identify and consolidate all the reported variants in WWOX to distinguish between disease-causing alleles and their associated severity, and benign variants, with the aim of improving diagnosis and increasing therapeutic efforts. Here, we provide a comprehensive review of the literature on WWOX, and analyze the pathogenic variants from published and unpublished reports by collecting entries from the ClinVar, DECIPHER, VarSome, and PubMed databases to generate the largest dataset of WWOX pathogenic variants. We estimate the correlation between variant type and patient phenotype, and delineate the impact of each variant, and used GnomAD to cross reference these variants found in the general population. From these searches, we generated the largest published cohort of WWOX in iduals. We conclude with a discussion on potential personalized medicine approaches to tackle the devastating disorders associated with WWOX mutations.
Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation
Publisher: Elsevier BV
Date: 11-2013
Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1038/MT.2012.164
Safety and efficacy of an engineered hepatotropic AAV gene therapy for ornithine transcarbamylase deficiency in cynomolgus monkeys
Publisher: Elsevier BV
Date: 12-2021
Selection and evaluation of clinically relevant AAV variants in a xenograft liver model
Publisher: Springer Science and Business Media LLC
Date: 25-12-2013
DOI: 10.1038/NATURE12875
Erythroid-specific Human Factor IX Delivery From In Vivo Selected Hematopoietic Stem Cells Following Nonmyeloablative Conditioning in Hemophilia B Mice
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1038/MT.2008.161
Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2
Publisher: Elsevier BV
Date: 06-2020
Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity
Publisher: Elsevier BV
Date: 2018
Second Generation Codon Optimized Minicircle (CoMiC) for Nonviral Reprogramming of Human Adult Fibroblasts
Publisher: Springer New York
Date: 2014
Age-Related Seroprevalence of Antibodies Against AAV-LK03 in a UK Population Cohort
Publisher: Mary Ann Liebert Inc
Date: 2019
DOI: 10.1089/HUM.2018.098
Erratum: Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa (Science Translational Medicine (2014) 6 (267er8))
Publisher: American Association for the Advancement of Science (AAAS)
Date: 17-12-2014
Molecular mechanisms of amyotrophic lateral sclerosis as broad therapeutic targets for gene therapy applications utilizing adeno‐associated viral vectors
Publisher: Wiley
Date: 14-02-2023
DOI: 10.1002/MED.21937
Abstract: Despite the devastating clinical outcome of the neurodegenerative disease, amyotrophic lateral sclerosis (ALS), its etiology remains mysterious. Approximately 90% of ALS is characterized as sporadic, signifying that the patient has no family history of the disease. The development of an impactful disease modifying therapy across the ALS spectrum has remained out of grasp, largely due to the poorly understood mechanisms of disease onset and progression. Currently, ALS is invariably fatal and rapidly progressive. It is hypothesized that multiple factors can lead to the development of ALS, however, treatments are often focused on targeting specific familial forms of the disease (10% of total cases). There is a strong need to develop disease modifying treatments for ALS that can be effective across the full ALS spectrum of familial and sporadic cases. Although the onset of disease varies significantly between patients, there are general disease mechanisms and progressions that can be seen broadly across ALS patients. Therefore, this review explores the targeting of these widespread disease mechanisms as possible areas for therapeutic intervention to treat ALS broadly. In particular, this review will focus on targeting mechanisms of defective protein homeostasis and RNA processing, which are both increasingly recognized as design principles of ALS pathogenesis. Additionally, this review will explore the benefits of gene therapy as an approach to treating ALS, specifically focusing on the use of adeno‐associated virus (AAV) as a vector for gene delivery to the CNS and recent advances in the field.
Adeno-associated virus serotypes for gene therapeutics
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.COPH.2015.07.006
Abstract: Gene transfer vectors based on adeno-associated virus (AAV) are showing exciting therapeutic promise in early phase clinical trials. The ability to cross-package the prototypic AAV2 vector genome into different capsids is a powerful way of conferring novel tropism and biology, with evolving capsid engineering technologies and directed evolution approaches further enhancing the utility and flexibility of these vectors. Novel properties of specific capsids show unpredictable species and cell-type specificity. Therefore, full realisation of the therapeutic potential of AAV vectors requires the development of more therapeutically predictive preclinical methods for evaluating capsid performance. This will strongly complement an iterative approach to the evaluation of capsid variants in the clinic and, should wherever possible, include the determination of gene transfer efficiencies.
Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation
Publisher: Cold Spring Harbor Laboratory
Date: 10-07-2023
DOI: 10.1101/2023.07.10.548299
Abstract: Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8-derived variants performed efficiently. This study establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.
Directed evolution of AAV capsids for improved efficacy and specificity of delivery to preclinical models of human liver
Publisher: Wojskowy Instytut Medyczny – Panstwowy Instytut Badawczy
Date: 31-03-2023
DOI: 10.53301/LW/156374
Abstract: Recombinant vectors derived from adeno-associated viruses (rAAVs) are the leading platform in human gene therapy applications, with high-profile ex les targeting diseases of the central nervous system, eye and liver. The liver, quite likely a natural host organ for wild type AAV2, is a particularly attractive target for the development of AAV-mediated gene therapies. Despite large number of AAV variants current at various stages of development as carriers of liver therapeutics, thus far no liver-directed AAV-based therapy has obtained market authorization. Strong preclinical data is the cornerstone of any translational program, and while AAV bioengineering is commonly applied to try to develop novel human-tropic vectors for clinical applications, due to species-to-species differences, dedicated vectors to support preclinical work may need to be developed. Here we applied AAV directed evolution and i in vitro /i selection to identify AAV capsids that target human liver cells i in vitro /i . Using DNA shuffling technology, we have generated a capsid gene library based on natural parental serotypes (AAV1 through AAV12). Shuffled capsid library was selected in a preclinical model of human liver. The AAV variants enriched based on their improved efficiency of transduction of a human hepatocyte cell line were vectorized and subsequently functionally characterized on human cell lines. This directed evolution method enabled us to select novel AAV variant, AAV-CH4.2. While the selected variant did not exceed the parental serotype in terms of transduction efficiency, it was substantially more efficient at packaging than its closest homolog, serotypes AAV6. Based on its strong transduction profile and manufacturability, we believe that AAV-CH4.2 is a strong candidate for further evaluation and as a potential novel gene therapy vector for preclinical studies in human liver applications.
Restoring the natural tropism of AAV2 vectors for human liver
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-09-2020
DOI: 10.1126/SCITRANSLMED.ABA3312
Abstract: AAVs isolated from human liver are tropic for human hepatocytes in vivo, but this phenotype becomes attenuated after passage in culture.
Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors
Publisher: Cold Spring Harbor Laboratory
Date: 30-09-2022
DOI: 10.1101/2022.09.28.510021
Abstract: The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.
Optogenetic approaches to vision restoration
Publisher: Elsevier BV
Date: 2019
DOI: 10.1016/J.EXER.2018.09.003
Abstract: Inherited retinal disease (IRD) affects about 1 in 3000 to 1 in 5000 in iduals and is now believed to be the most common cause of blindness registration in developed countries. Until recently, the management of such conditions had been exclusively supportive. However, advances in molecular biology and medical engineering have now seen the rise of a variety of approaches to restore vision in patients with IRDs. Optogenetic approaches are primarily aimed at rendering secondary and tertiary neurons of the retina light-sensitive in order to replace degenerate or dysfunctional photoreceptors. Such approaches are attractive because they provide a "causative gene-independent" strategy, which may prove suitable for a variety of patients with IRD. We discuss theoretical and practical considerations in the selection of optogenetic molecules, vectors, surgical approaches and review previous trials of optogenetics for vision restoration. Optogenetic approaches to vision restoration have yielded promising results in pre-clinical trials and a phase I/II clinical trial is currently underway (ClinicalTrials.gov NCT02556736). Despite the significant inroads made in recent years, the ideal optogenetic molecule, vector and surgical approach have yet to be established.
Related Organisations
The Salk Institute For Biological Studies
Location: United States of America
University Of California San Diego Rady School Of Management
Location: United States of America
Weill Cornell Graduate School Of Medical Sciences
Location: United States of America
Related Funding Activities
No related grants have been discovered for Leszek Lisowski.