Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy

ABSTRACT Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.

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Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

Thrombosis and Haemostasis ◽

10.1160/th13-02-0154 ◽

2013 ◽

Vol 110 (08) ◽

pp. 244-256 ◽

Cited By ~ 20

Author(s):

Chung-Yang Kao ◽

Shu-Jhu Yang ◽

Mi-Hua Tao ◽

Yung-Ming Jeng ◽

I-Shing Yu ◽

...

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Therapeutic Potential ◽

Factor Ix ◽

Adeno Associated Virus ◽

Viral Dose ◽

Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

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Utility of microminipigs for evaluating liver-mediated gene expression in the presence of neutralizing antibody against vector capsid

Gene Therapy ◽

10.1038/s41434-020-0125-0 ◽

2020 ◽

Vol 27 (9) ◽

pp. 427-434

Author(s):

Ryota Watano ◽

Tsukasa Ohmori ◽

Shuji Hishikawa ◽

Asuka Sakata ◽

Hiroaki Mizukami

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Transgene Expression ◽

Imaging System ◽

Standard Dose ◽

Neutralizing Antibody ◽

Aav Vector ◽

Adeno Associated Virus ◽

In Vivo Imaging System

Abstract Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

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Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721883115 ◽

2018 ◽

Vol 115 (15) ◽

pp. E3529-E3538 ◽

Cited By ~ 5

Author(s):

Sarah Smith-Moore ◽

Stuart J. D. Neil ◽

Cornel Fraefel ◽

R. Michael Linden ◽

Mathieu Bollen ◽

...

Keyword(s):

Gene Therapy ◽

Helper Virus ◽

Protein Phosphatase 1 ◽

Wild Type ◽

Fha Domain ◽

Adeno Associated Virus ◽

Histone 3 ◽

Rep Proteins ◽

Cellular Factors

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)–PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

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Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.677895 ◽

2021 ◽

Vol 14 ◽

Author(s):

Emilie Audouard ◽

Valentin Oger ◽

Béatrix Meha ◽

Nathalie Cartier ◽

Caroline Sevin ◽

...

Keyword(s):

Spinal Cord ◽

Gene Therapy ◽

Metachromatic Leukodystrophy ◽

Early Death ◽

Lysosomal Storage ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Brain And Spinal Cord ◽

Sulfatide Accumulation

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.

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In Vivo Potency Assay for Adeno-Associated Virus–Based Gene Therapy Vectors Using AAVrh.10 as an Example

Human Gene Therapy Methods ◽

10.1089/hgtb.2017.246 ◽

2018 ◽

Vol 29 (3) ◽

pp. 146-155 ◽

Cited By ~ 5

Author(s):

Bishnu P. De ◽

Alvin Chen ◽

Christiana O. Salami ◽

Benjamin Van de Graaf ◽

Jonathan B. Rosenberg ◽

...

Keyword(s):

Gene Therapy ◽

Potency Assay ◽

Adeno Associated Virus ◽

Gene Therapy Vectors

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Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy

Biological Chemistry ◽

10.1515/bc.1999.078 ◽

1999 ◽

Vol 380 (6) ◽

Cited By ~ 63

Author(s):

H. Büeler

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Gene Delivery ◽

Viral Vectors ◽

Delivery Systems ◽

Adeno Associated Virus ◽

Viral Genes ◽

Virus Recombinant

AbstractAdeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression

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Nanotechnology and adeno-associated virus-based decorin gene therapy ameliorates peritoneal fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00653.2013 ◽

2014 ◽

Vol 307 (7) ◽

pp. F777-F782 ◽

Cited By ~ 16

Author(s):

Kunal Chaudhary ◽

Harold Moore ◽

Ashish Tandon ◽

Suneel Gupta ◽

Ramesh Khanna ◽

...

Keyword(s):

Gene Therapy ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Mesothelial Cell ◽

Peritoneal Fibrosis ◽

Tissue Samples ◽

Adeno Associated Virus ◽

Peritoneal Tissue ◽

Stage Renal Disease

Peritoneal dialysis (PD) is a life-sustaining therapy for end-stage renal disease (ESRD), used by 10–15% of the dialysis population worldwide. Peritoneal fibrosis (PF) is a known complication of long-term PD and frequently follows episodes of peritonitis, rendering the peritoneal membrane inadequate for dialysis. Transforming growth factor (TGF)-β is an inducer of fibrosis in several tissues and organs, and its overexpression has been correlated with PF. Animal models of peritonitis have shown an increase in expression of TGF-β in the peritoneal tissue. Decorin, a proteoglycan and component of the extracellular matrix, inactivates TGF-β, consequently reducing fibrosis in many tissues. Recently, gold nanoparticles (GNP) have been used for drug delivery in a variety of settings. In the present study, we tested the possibility that GNP-delivered decorin gene therapy ameliorates zymosan-mediated PF. We created a PF model using zymosan-induced peritonitis. Rats were treated with no decorin, GNP-decorin, or adeno-associated virus-decorin (AAV-decorin) and compared with controls. Tissue samples were then stained for Masson's trichrome, enface silver, and hematoxylin and eosin, and immunohistochemistry was carried out with antibodies to TGF-β1, α-smooth muscle actin (α-SMA), and VEGF. Animals which were treated with GNP-decorin and AAV-decorin gene therapy had significant reductions in PF compared with untreated animals. Compared with untreated animals, the treated animals had better preserved peritoneal mesothelial cell size, a significant decrease in peritoneal thickness, and decreased α-SMA. Quantitative PCR measurements showed a significant decrease in the peritoneal tissue levels of α-SMA, TGF-β, and VEGF in treated vs. untreated animals. This study shows that both GNP-delivered and AAV-mediated decorin gene therapies significantly decrease PF in vivo in a rodent model. This approach has important clinical translational potential in providing a therapeutic strategy to prevent PF in PD patients.

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An experimental study to determine and correlate choline acetyltransferase assay with functional muscle testing after nerve injury

Journal of Neurosurgery ◽

10.3171/2014.1.jns122241 ◽

2014 ◽

Vol 120 (5) ◽

pp. 1125-1130 ◽

Cited By ~ 1

Author(s):

Torpon Vathana ◽

Tim H. J. Nijhuis ◽

Patricia F. Friedrich ◽

Allen T. Bishop ◽

Alexander Y. Shin

Keyword(s):

Tibialis Anterior ◽

Peroneal Nerve ◽

Force Measurement ◽

Tibialis Anterior Muscle ◽

Muscle Weight ◽

Tetanic Force ◽

Chat Activity ◽

Significant Difference ◽

Group 3 ◽

Group 2

Object Choline acetyltransferase (ChAT) is an enzyme synthesized within the body of a motor neuron whose role is to form the neurotransmitter acetylcholine. Quantification of ChAT levels in motor or mixed nerves has been proposed to provide information regarding the viability of a proximal nerve stump for motor neurotization following brachial plexus injury. To do so requires information regarding normal ChAT levels and those in injured nerves, as well as the correlation of ChAT level determined at surgery with eventual motor recovery. The purpose of this study was to determine ChAT activity in the normal and injured sciatic/peroneal nerve in a rat model, evaluate the correlation between ChAT and motor recovery, find the relationship between ChAT activity and isometric muscle force, and elucidate the parallel between ChAT activity and acetylcholinesterase (AChE) activity. Methods Sixty animals were divided into 3 groups. The sciatic nerves in Group 1 were transected without repair. Nerves in Group 2 were transected and repaired. Nerves in Group 3 sustained a crush injury followed by transection and reconstruction. All animals were allowed 12 weeks of recovery followed by evaluation of ChAT levels in the peroneal nerve, correlated with measures of maximal isometric tibialis anterior muscle force and muscle weight (the operated side normalized to the control side). Karnovsky AChE staining of peroneal nerve segments was also compared with radiochemical assay of ChAT activity in the same nerve. Results A significant difference in the tibialis anterior isometric tetanic force and the tibialis anterior muscle weight index (TAMI) was noted between Group 1 and Groups 2 and 3 (p < 0.0001); no significant difference was found comparing Group 2 with Group 3. The correlation between the force measurement and the TAMI was 0.382. Both AChE measurement and ChAT activity demonstrated significantly fewer fibers in the operated nerve compared with the contralateral nerve. Intergroup variability could also be illustrated using these tests. The correlation coefficient between the isometric tetanic force measurement and the ChAT analysis in Groups 1 and 2 was 0.468. The correlation for the AChE staining and the isometric tetanic force measurement was 0.111. The correlation between the TAMI and the ChAT levels was 0.773. The correlation between the TAMI and the AChE-stained fibers was 0.640. Correlating AChE staining to the ChAT analysis produced a correlation of 0.712. Conclusions The great variability in all groups and weak correlations to the functional muscle assessments and the ChAT radiochemical assay made this technique an unreliable method of determining motor nerve viability.

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Lack of Repeat Transduction by Recombinant Adeno-Associated Virus Type 5/5 Vectors in the Mouse Airway

Journal of Virology ◽

10.1128/jvi.01010-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12360-12367 ◽

Cited By ~ 21

Author(s):

Stephanie G. Sumner-Jones ◽

Deborah R. Gill ◽

Stephen C. Hyde

Keyword(s):

Transgene Expression ◽

Lung Diseases ◽

Repeated Administration ◽

Transduction Efficiency ◽

Viral Gene ◽

Rapid Rise ◽

Adeno Associated Virus ◽

Subsequent Failure

ABSTRACT While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.

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The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

Journal of Virology ◽

10.1128/jvi.00878-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9831-9836 ◽

Cited By ~ 264

Author(s):

Bassel Akache ◽

Dirk Grimm ◽

Kusum Pandey ◽

Stephen R. Yant ◽

Hui Xu ◽

...

Keyword(s):

Gene Therapy ◽

Cultured Cells ◽

Human Gene ◽

Laminin Receptor ◽

Transduction Efficiency ◽

Adeno Associated Virus ◽

Virus Serotype ◽

Tumor Gene

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

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