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

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

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Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors

Journal of Pharmaceutical Sciences ◽

10.1016/j.xphs.2021.06.010 ◽

2021 ◽

Author(s):

Jared S. Bee ◽

Kristin O'Berry ◽

Yu (Zoe) Zhang ◽

Megan Kuhn Phillippi ◽

Akanksha Kaushal ◽

...

Keyword(s):

Gene Therapy ◽

Adeno Associated Virus ◽

Virus Gene ◽

Gene Therapy Vectors

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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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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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Identification and Validation of Small Molecules That Enhance Recombinant Adeno-associated Virus Transduction following High-Throughput Screens

Journal of Virology ◽

10.1128/jvi.02953-15 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7019-7031 ◽

Cited By ~ 22

Author(s):

Sarah C. Nicolson ◽

Chengwen Li ◽

Matthew L. Hirsch ◽

Vincent Setola ◽

R. Jude Samulski

Keyword(s):

Gene Therapy ◽

Gene Delivery ◽

High Throughput ◽

Adeno Associated Virus ◽

Virus Gene ◽

Diploid Cells ◽

High Throughput Screens ◽

Group 1

ABSTRACTWhile the recent success of adeno-associated virus (AAV)-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2,396 compounds. We identified 13 compounds that were capable of enhancing transduction, of which 12 demonstrated vector-specific effects and 1 could also enhance vector-independent transgene expression. Many of these compounds had similar properties and could be categorized into five groups: epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors.In vivoanalysis of rAAV2 and all of the clinically relevant compounds revealed that, consistent with ourin vitroresults, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectorsin vivousing an AAV8 capsid that is known to exhibit robust liver tropism. Consistent with ourin vitroresults, teniposide coadministration greatly enhanced fragmented rAAV8 transduction at 48 h and 8 days. This study provides a foundation based on the rAAV small-molecule screen methodology, which is ideally used for more-diverse libraries of compounds that can be tested for potentiating rAAV transduction.IMPORTANCEThis study seeks to enhance the capability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs were tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50 to 100 times better with than without teniposide, a compound that is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy, which could result in safe and effective treatments for diverse acquired or genetic diseases.

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A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase ϕC31 and the Sleeping Beauty transposase

Molecular Therapy ◽

10.1016/j.ymthe.2005.01.010 ◽

2005 ◽

Vol 11 (5) ◽

pp. 695-706 ◽

Cited By ~ 51

Author(s):

Anja Ehrhardt ◽

Hui Xu ◽

Zan Huang ◽

Jeffrey A. Engler ◽

Mark A. Kay

Keyword(s):

Gene Therapy ◽

Sleeping Beauty ◽

In Vivo Evaluation ◽

Nonviral Gene Therapy ◽

Gene Therapy Vectors

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The packaging capacity of adeno-associated virus (AAV) and the potential forwild-type-plusAAV gene therapy vectors

FEBS Letters ◽

10.1016/s0014-5793(97)00311-6 ◽

1997 ◽

Vol 407 (1) ◽

pp. 78-84 ◽

Cited By ~ 57

Author(s):

Paul L. Hermonat ◽

J.Gerald Quirk ◽

Brian M. Bishop ◽

Li Han

Keyword(s):

Gene Therapy ◽

Adeno Associated Virus ◽

Gene Therapy Vectors ◽

Packaging Capacity

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Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

Molecular Therapy ◽

10.1038/mt.2014.89 ◽

2014 ◽

Vol 22 (8) ◽

pp. 1484-1493 ◽

Cited By ~ 51

Author(s):

Benjamin S Schuster ◽

Anthony J Kim ◽

Joshua C Kays ◽

Mia M Kanzawa ◽

William B Guggino ◽

...

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Adeno Associated Virus ◽

Virus Gene ◽

Cystic Fibrosis Sputum ◽

Gene Therapy Vectors

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Weighing the DNA content of Adeno-Associated Virus vectors with zeptogram precision using nanomechanical resonators

10.1101/2021.11.15.468734 ◽

2021 ◽

Author(s):

Georgios Katsikis ◽

Iris E Hwang ◽

Wade Wang ◽

Vikas S Bhat ◽

Nicole L McIntosh ◽

...

Keyword(s):

Gene Therapy ◽

Quality Control ◽

Viral Vectors ◽

Analytical Ultracentrifugation ◽

Vaccine Development ◽

Turnaround Time ◽

Charge Detection ◽

Adeno Associated Virus ◽

Nanomechanical Resonators

Quantifying the composition of viral vectors used in vaccine development and gene therapy is critical for assessing their functionality. Adeno-Associated Virus (AAV) vectors, which are the most widely used viral vectors for in-vivo gene therapy, are typically characterized using PCR, ELISA, and Analytical Ultracentrifugation which require laborious protocols or hours of turnaround time. Emerging methods such as Charge-Detection Mass Spectroscopy, Static Light Scattering, and Mass Photometry offer turnaround times of minutes for measuring AAV mass, but mostly require purified AAV-based reference materials for calibration. Here, we demonstrate a method for using Suspended Nanomechanical Resonators (SNR) to directly measure both AAV mass and aggregation from a few microliters of sample within minutes. We achieve a resolution near 10 zeptograms which corresponds to 1% of the genome holding capacity of the AAV capsid. Our results show the potential of our method for providing real-time quality control of viral vectors during biomanufacturing.

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