Publications by authors named "Sharon C Cunningham"

20 Publications

  • Page 1 of 1

Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice.

Cells 2020 10 2;9(10). Epub 2020 Oct 2.

School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia.

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin () to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver - ± the β-TF to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/ that results in transgene integration were used. Diabetic mice that received AAV8- became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8--FUR and AAV8- and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/-/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/ system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9102227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600325PMC
October 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhepr.2019.100065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005564PMC
February 2020

Liver-Targeted Angiotensin Converting Enzyme 2 Therapy Inhibits Chronic Biliary Fibrosis in Multiple Drug-Resistant Gene 2-Knockout Mice.

Hepatol Commun 2019 Dec 10;3(12):1656-1673. Epub 2019 Oct 10.

Department of Medicine University of Melbourne Austin Health Heidelberg Australia.

There is a large unmet need for effective therapies for cholestatic disorders, including primary sclerosing cholangitis (PSC), a disease that commonly results in liver failure. Angiotensin (Ang) II of the renin Ang system (RAS) is a potent profibrotic peptide, and Ang converting enzyme 2 (ACE2) of the alternate RAS breaks down Ang II to antifibrotic peptide Ang-(1-7). In the present study, we investigated long-term effects of ACE2 delivered by an adeno-associated viral vector and short-term effects of Ang-(1-7) peptide in multiple drug-resistant gene 2-knockout (Mdr2-KO) mice. These mice develop progressive biliary fibrosis with pathologic features closely resembling those observed in PSC. A single intraperitoneal injection of ACE2 therapy markedly reduced liver injury ( < 0.05) and biliary fibrosis ( < 0.01) at both established (3-6 months of age) and advanced (7-9 months of age) disease compared to control vector-injected Mdr2-KO mice. This was accompanied by increased hepatic Ang-(1-7) levels ( < 0.05) with concomitant reduction in hepatic Ang II levels ( < 0.05) compared to controls. Moreover, Ang-(1-7) peptide infusion improved liver injury ( < 0.05) and biliary fibrosis ( < 0.0001) compared to saline-infused disease controls. The therapeutic effects of both ACE2 therapy and Ang-(1-7) infusion were associated with significant ( < 0.01) reduction in hepatic stellate cell (HSC) activation and collagen expression. While ACE2 therapy prevented the loss of epithelial characteristics of hepatocytes and/or cholangiocytes , Ang-(1-7) prevented transdifferentiation of human cholangiocytes (H69 cells) into the collagen-secreting myofibroblastic phenotype . We showed that an increased ratio of hepatic Ang-(1-7) to Ang II levels by ACE2 therapy results in the inhibition of HSC activation and biliary fibrosis. ACE2 therapy has the potential to treat patients with biliary diseases, such as PSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep4.1434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887688PMC
December 2019

Insights into Gene Therapy for Urea Cycle Defects by Mathematical Modeling.

Hum Gene Ther 2019 11 17;30(11):1385-1394. Epub 2019 Jul 17.

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

Metabolic liver diseases are attractive gene therapy targets that necessitate reconstitution of enzymatic activity in functionally complex biochemical pathways. The levels of enzyme activity required in individual hepatocytes and the proportion of the hepatic cell mass that must be gene corrected for therapeutic benefit vary in a disease-dependent manner that is difficult to predict. While empirical evaluation is inevitably required, useful insights can nevertheless be gained from knowledge of disease pathophysiology and theoretical approaches such as mathematical modeling. Urea cycle defects provide an excellent example. Building on a previously described one-compartment model of the urea cycle, we have constructed a two-compartment model that can simulate liver-targeted gene therapy interventions using the computational program . The model predicts that therapeutically effective reconstitution of ureagenesis will correlate most strongly with the proportion of the hepatic cell mass transduced rather than the level of enzyme-encoding transgene expression achieved in individual hepatocytes. Importantly, these predictions are supported by experimental data in mice and human genotype/phenotype correlations. The most notable example of the latter is ornithine transcarbamylase deficiency (X-linked) where impairment of ureagenesis in male and female patients is closely simulated by the one- and two-compartment models, respectively. Collectively, these observations support the practical value of mathematical modeling in evaluation of the disease-specific gene transfer challenges posed by complex metabolic phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2019.053DOI Listing
November 2019

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV-piggyBac Gene Therapy.

Hepatology 2019 12 26;70(6):2047-2061. Epub 2019 Jun 26.

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.

Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4 mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4 mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.30773DOI Listing
December 2019

AAV-Mediated Gene Delivery to the Mouse Liver.

Methods Mol Biol 2019 ;1937:213-219

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

The liver is an attractive target for gene therapy due to the high incidence of liver disease phenotypes. Adeno-associated viral vectors (AAV) are currently the most popular gene delivery system for targeting the liver, reflecting high transduction efficiency in vivo and the availability of a toolkit of multiple different capsids with high liver tropism. While AAV vectors confer stable gene transfer in the relatively quiescent adult liver, the predominantly episomal nature of AAV vector genomes results in less stable expression in the growing liver as a consequence of episome clearance during hepatocellular replication. This is an important consideration in experimental design involving young animals, particularly mice, where liver growth is rapid. Given the immense value of murine models for dissecting disease pathophysiology, experimental therapeutics and vector development, this technical manuscript focuses on AAV-mediated transduction of the mouse liver. Xenograft models, in which chimeric mouse-human livers can be established, are also amenable to AAV-mediated gene transfer and have proven to be powerful tools for in vivo selection and characterization of novel human-specific capsids. While yet to be confirmed, such models have the potential to more accurately predict transduction efficiency of clinical candidate vectors than nonhuman primate models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-9065-8_12DOI Listing
July 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.3893DOI Listing
August 2017

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

Hepatology 2015 Aug 23;62(2):417-28. Epub 2015 May 23.

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

Unlabelled: Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data.

Conclusion: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.27842DOI Listing
August 2015

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice.

Mol Ther 2015 Sep 25;23(9):1434-43. Epub 2015 May 25.

Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, Victoria, Australia.

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1-7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1-7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2015.92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817885PMC
September 2015

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Nature 2014 Feb 25;506(7488):382-6. Epub 2013 Dec 25.

Stanford University, School of Medicine, Departments of Pediatrics and Genetics, 269 Campus Drive, Stanford, California 94305, USA.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature12875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939040PMC
February 2014

Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice.

Mol Ther 2013 Oct 2;21(10):1823-31. Epub 2013 Jul 2.

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

Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2013.139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808136PMC
October 2013

Gene therapy for metabolic disorders: an overview with a focus on urea cycle disorders.

J Inherit Metab Dis 2012 Jul 9;35(4):641-5. Epub 2012 Mar 9.

Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.

Many metabolic diseases are compelling candidates for gene therapy, and are the subject of vigorous pre-clinical research. Successful phenotype correction in mouse models is now commonplace and research effort is increasingly being directed towards addressing the translational challenges inherent in human clinical trials. This paper places current efforts to develop gene therapy approaches to metabolic disease in historical context and describes contemporary research in the authors' laboratory on urea cycle defects, particularly ornithine transcarbamylase deficiency, in a manner that is illustrative of the general state of the field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10545-012-9467-0DOI Listing
July 2012

Induction and prevention of severe hyperammonemia in the spfash mouse model of ornithine transcarbamylase deficiency using shRNA and rAAV-mediated gene delivery.

Mol Ther 2011 May 8;19(5):854-9. Epub 2011 Mar 8.

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

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2011.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098641PMC
May 2011

Sexually dimorphic patterns of episomal rAAV genome persistence in the adult mouse liver and correlation with hepatocellular proliferation.

Mol Ther 2009 Sep 30;17(9):1548-54. Epub 2009 Jun 30.

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

Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2009.139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835267PMC
September 2009

AAV2/8-mediated correction of OTC deficiency is robust in adult but not neonatal Spf(ash) mice.

Mol Ther 2009 Aug 21;17(8):1340-6. Epub 2009 Apr 21.

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

Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2009.88DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835243PMC
August 2009

Gene Delivery to the Juvenile Mouse Liver Using AAV2/8 Vectors.

Mol Ther 2008 Jun 8;16(6):1081-1088. Epub 2016 Dec 8.

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

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (IP) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2008.72DOI Listing
June 2008

Gene delivery to the juvenile mouse liver using AAV2/8 vectors.

Mol Ther 2008 Jun 15;16(6):1081-8. Epub 2008 Apr 15.

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

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (i.p.) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2008.72DOI Listing
June 2008

AAV vectors encoding malarial antigens stimulate antigen-specific immunity but do not protect from parasite infection.

Vaccine 2007 Jan 9;25(6):1014-22. Epub 2006 Oct 9.

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

This study explores the utility of recombinant adeno-associated virus (rAAV) as a genetic vaccine delivery system using muscle as a target tissue. A single injection of rAAV encoding the malarial antigens MSP4 (Plasmodium falciparum) or MSP4/5 (Plasmodium yoelii) stimulated long-term antigen-specific antibody responses. Anti-MSP4/5 immunity stimulated by AAV was not protective against P. yoelii infection and efforts taken to augment antibody responses against MSP4/5, either by priming with plasmid DNA or AAV and boosting with rAAV were unsuccessful. Alternative strategies such as inclusion of genetic adjuvants into the AAV vector will be necessary to stimulate an adequate level of anti-malarial protective immunity in this model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2006.09.072DOI Listing
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5694/j.1326-5377.2005.tb06785.xDOI Listing
May 2005
-->