Publications by authors named "Leszek Lisowski"

44 Publications

Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids.

Mol Ther Methods Clin Dev 2021 Jun 24;21:607-620. Epub 2021 Apr 24.

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

Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model.
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http://dx.doi.org/10.1016/j.omtm.2021.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142051PMC
June 2021

Neurological Disorders Associated with WWOX Germline Mutations-A Comprehensive Overview.

Cells 2021 Apr 7;10(4). Epub 2021 Apr 7.

The Concern Foundation Laboratories, The Lautenberg Center for Immunology and Cancer Research, Department of Immunology and Cancer Research-IMRIC, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

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). Individuals 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, hampering 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 individuals. We conclude with a discussion on potential personalized medicine approaches to tackle the devastating disorders associated with WWOX mutations.
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http://dx.doi.org/10.3390/cells10040824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067556PMC
April 2021

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation.

Xenotransplantation 2021 05 14;28(3):e12669. Epub 2020 Dec 14.

Immunology Department, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

Background: 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.

Methods: 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.

Results: 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 (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation.

Conclusion: 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.
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http://dx.doi.org/10.1111/xen.12669DOI Listing
May 2021

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

Gastroenterology 2021 03 14;160(4):1224-1239.e30. Epub 2020 Nov 14.

Herbert Irving Comprehensive Cancer Center, Division of Digestive and Liver Diseases, Department of Medicine, Columbia University, New York, New York.

Background & Aims: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered 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.

Methods: 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.

Results: 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 intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis.

Conclusions: 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.
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http://dx.doi.org/10.1053/j.gastro.2020.11.011DOI Listing
March 2021

Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.

Nat Commun 2020 09 25;11(1):4871. Epub 2020 Sep 25.

Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Tasmania, Australia.

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.
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http://dx.doi.org/10.1038/s41467-020-18715-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519688PMC
September 2020

A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Angiogenesis 2021 02 15;24(1):97-110. Epub 2020 Sep 15.

Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
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http://dx.doi.org/10.1007/s10456-020-09745-7DOI Listing
February 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

Attenuation of Heparan Sulfate Proteoglycan Binding Enhances Transduction of Human Primary Hepatocytes with AAV2.

Mol Ther Methods Clin Dev 2020 Jun 13;17:1139-1154. Epub 2020 May 13.

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

Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes , an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional . Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency . The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.
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http://dx.doi.org/10.1016/j.omtm.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260615PMC
June 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

High-Throughput , and Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction.

Hum Gene Ther 2020 05 26;31(9-10):575-589. Epub 2020 Feb 26.

Translational Vectorology Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, Australia.

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with , and applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells as well as transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.
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http://dx.doi.org/10.1089/hum.2019.264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232709PMC
May 2020

A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus.

Hum Gene Ther Methods 2019 12;30(6):206-213

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

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 -acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.
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http://dx.doi.org/10.1089/hgtb.2019.276DOI Listing
December 2019

Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton.

Mol Ther Methods Clin Dev 2019 Dec 5;15:101-111. Epub 2019 Sep 5.

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

A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in mouse models.
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http://dx.doi.org/10.1016/j.omtm.2019.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804917PMC
December 2019

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease.

Hum Mol Genet 2020 01;29(1):20-30

Harry Perkins Institute of Medical Research, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.
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http://dx.doi.org/10.1093/hmg/ddz214DOI Listing
January 2020

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

Directed Evolution of Adeno-Associated Virus Vectors in Human Cardiomyocytes for Cardiac Gene Therapy.

Heart Lung Circ 2018 Nov;27(11):1270-1273

Center for Heart Research, The Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia; Department of Cardiology, Westmead Hospital, Sydney, NSW, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.hlc.2018.08.014DOI Listing
November 2018

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

Cell 2018 10 13;175(2):530-543.e24. Epub 2018 Sep 13.

Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW 2050, Australia; Discipline of Dermatology, Faculty of Medicine and Health, University of Sydney, NSW 2006, Australia; Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; Department of Dermatology, Medical University of Vienna, Vienna 1090, Austria. Electronic address:

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
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http://dx.doi.org/10.1016/j.cell.2018.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800251PMC
October 2018

Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle.

Mol Ther Methods Clin Dev 2018 Sep 24;10:144-155. Epub 2018 Jul 24.

Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants and in various primary and immortalized myogenic lines . Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.
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http://dx.doi.org/10.1016/j.omtm.2018.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077147PMC
September 2018

Age-Related Seroprevalence of Antibodies Against AAV-LK03 in a UK Population Cohort.

Hum Gene Ther 2019 01 19;30(1):79-87. Epub 2018 Oct 19.

1 Genetics and Genomic Medicine Programme, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapy. The presence of neutralizing antibodies (Nab) against AAV capsids decreases cell transduction efficiency and is a common exclusion criterion for participation in clinical trials. Novel engineered capsids are being generated to improve gene delivery to the target cells and facilitate success of clinical trials; however, the prevalence of antibodies against such capsids remains largely unknown. We therefore assessed the seroprevalence of antibodies against a novel synthetic liver-tropic capsid AAV-LK03. We measured seroprevalence of immunoglobulin (Ig)G (i.e., neutralizing and nonneutralizing) antibodies and Nab to AAV-LK03 in a cohort of 323 UK patients (including 260 pediatric) and 52 juvenile rhesus macaques. We also performed comparative analysis of seroprevalence of Nab against wild-type AAV8 and AAV3B capsids. Overall IgG seroprevalence for AAV-LK03 was 39% in human samples. The titer increased with age. Prevalence of Nab was 23%, 35%, and 18% for AAV-LK03, AAV3B, and AAV8, respectively, with the lowest seroprevalence between 3 and 17 years of age for all serotypes. Presence of Nab against AAV-LK03 decreased from 36% in the youngest cohort (birth to 6 months) to 7% in older primary school-age children (9-11 years) and then progressively increased to 54% in late adulthood. Cross-reactivity between serotypes was >60%. Nab seroprevalence in macaques was 62%, 85%, and 40% for AAV-LK03, AAV3B, and AAV8, respectively. When planning for AAV gene therapy clinical trials, knowing the seropositivity of the target population is critical. In the population studied, AAV seroprevalence for AAV serotypes tested was low. However, high cross-reactivity between AAV serotypes remains a barrier for re-injection. Shifts in Nab seroprevalence during the first decade need to be confirmed by longitudinal studies. This possibility suggests that pediatric patients could respond differently to AAV therapy according to age. If late childhood is an ideal age window, intervention at an early age when maternal Nab levels are high may be challenging. Nab-positive children excluded from trials could be rescreened for eligibility at regular intervals because this status may change.
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http://dx.doi.org/10.1089/hum.2018.098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343184PMC
January 2019

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

Mol Ther 2018 07 25;26(7):1818-1827. Epub 2018 Apr 25.

Department of Bioengineering, University of California, San Diego, San Diego, CA, USA. Electronic address:

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.
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http://dx.doi.org/10.1016/j.ymthe.2018.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035733PMC
July 2018

Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity.

Nat Commun 2017 12 12;8(1):2053. Epub 2017 Dec 12.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

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 alleles from bulk lung and pancreas reveals surprising diversity 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.
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http://dx.doi.org/10.1038/s41467-017-01519-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727199PMC
December 2017

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Mol Ther 2018 01 25;26(1):289-303. Epub 2017 Sep 25.

Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.
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http://dx.doi.org/10.1016/j.ymthe.2017.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763027PMC
January 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

RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice.

Nat Med 2016 05 11;22(5):557-62. Epub 2016 Apr 11.

Department of Pediatrics, Stanford University, Stanford, California, USA.

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.
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http://dx.doi.org/10.1038/nm.4079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860119PMC
May 2016

Adeno-associated virus serotypes for gene therapeutics.

Curr Opin Pharmacol 2015 Oct 25;24:59-67. Epub 2015 Aug 25.

Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Locked Bag 4001, Westmead 2145, NSW, Australia; Discipline of Paediatrics and Child Health, The University of Sydney, NSW, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.coph.2015.07.006DOI Listing
October 2015

Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.

Sci Transl Med 2014 Nov;6(264):264ra163

Program in Epithelial Biology, Department of Dermatology, Stanford University, Stanford, CA 94305, USA.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.
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http://dx.doi.org/10.1126/scitranslmed.3009540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428910PMC
November 2014

Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing.

J Am Coll Cardiol 2014 Aug;64(5):451-9

Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California. Electronic address:

Background: Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control.

Objectives: This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control.

Methods: The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology.

Results: Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future.

Conclusions: Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.
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http://dx.doi.org/10.1016/j.jacc.2014.04.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149735PMC
August 2014

Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

Methods Mol Biol 2014 ;1181:1-13

Lorry I. Lokey Stem Cell Research Building, Stanford University School of Medicine, 265 Campus Drive, Room G1105, Stanford, CA, 94305-5454, USA.

The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.
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http://dx.doi.org/10.1007/978-1-4939-1047-2_1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385867PMC
March 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.
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http://dx.doi.org/10.1038/nature12875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939040PMC
February 2014

Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.

Mol Ther 2014 Apr 6;22(4):725-33. Epub 2014 Jan 6.

Program in Epithelial Biology, Stanford University, School of Medicine, Stanford, California, USA.

Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.
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http://dx.doi.org/10.1038/mt.2013.290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982486PMC
April 2014
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