Preexisting Neutralizing Antibodies to Adeno-Associated Virus Capsids in Large Animals Other Than Monkeys May Confound In Vivo Gene Therapy Studies

ABSTRACT The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.

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Genetic Engineering of AAV Capsid Gene for Gene Therapy Application

Current Gene Therapy ◽

10.2174/1566523220666200930105521 ◽

2020 ◽

Vol 20 (5) ◽

pp. 321-332

Author(s):

Yunbo Liu ◽

Xu Zhang ◽

Lin Yang

Keyword(s):

Gene Therapy ◽

Neutralizing Antibodies ◽

Human Subjects ◽

Genetic Diseases ◽

Capsid Gene ◽

Human Populations ◽

Stable Gene ◽

Efficient Gene ◽

Gene Therapy Application

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

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Adeno-Associated Virus Neutralizing Antibodies in Large Animals and Their Impact on Brain Intraparenchymal Gene Transfer

Molecular Therapy — Methods & Clinical Development ◽

10.1016/j.omtm.2018.09.003 ◽

2018 ◽

Vol 11 ◽

pp. 65-72 ◽

Cited By ~ 8

Author(s):

Dan Wang ◽

Li Zhong ◽

Mengxin Li ◽

Jia Li ◽

Karen Tran ◽

...

Keyword(s):

Gene Transfer ◽

Neutralizing Antibodies ◽

Adeno Associated Virus ◽

Large Animals

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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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Abstract MP165: Exosome-mediated Encapsulation Alters AAV Antigenicity and Infectivity: Implications for Gene Delivery in the Heart

Circulation Research ◽

10.1161/res.127.suppl_1.mp165 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Marta Adamiak ◽

Yaxuan Liang ◽

Cherrie Sherman ◽

Shweta Lodha ◽

Erik Kohlbrenner ◽

...

Keyword(s):

Gene Therapy ◽

Gene Delivery ◽

Cardiac Function ◽

Neutralizing Antibodies ◽

Firefly Luciferase ◽

Interferometric Imaging ◽

Human Cardiomyocytes ◽

Clinical Benefits

Gene therapy is a promising approach for the treatment of cardiovascular disease. Current strategies for myocardial gene transfer include the use of adeno-associated virus (AAV) vectors. However, AAVs may not be ideal for gene therapy vectors owing to pre-existing AAV capsid immunity in the human population that may reduce transduction efficacy and hinder preclinical-to-clinical translation. Interestingly, recent studies suggest that exosome-mediated encapsulation may protect viruses from neutralizing antibodies (NAbs) against the capsid and promote viral infectivity. Here, we describe the ability of exosome-enveloped AAVs, i.e. exosomal AAVs (eAAVs), to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to purifiy eAAVs from AAV-producing HEK-293T cells, and used electron/confocal microscopy, qPCR, immunoblotting, dynamic light scattering and interferometric imaging measurements to characterize eAAV morphology, contents and mechanism of action. We confirmed eAAVs represent vesicular fractions that exhibit common exosome phenotype, along with the presence of virus particles, and demonstrated that eAAV infectious entry potentially involves trafficking via endocytic compartments. Using flow cytometry, Langendorff perfusion system and bioluminescence imaging, we then evaluated efficiency of heart targeting for eAAV9/eAAV6 and standard AAV9/AAV6 encoding for mCherry or firefly luciferase in human cardiomyocytes in vitro and in mouse model in vivo . Regardless of the presence or absence of NAbs, we showed that eAAVs are more efficient in transduction in the same titer ranges as compared to standard AAVs. To test therapeutic efficacy, we intramyocardially injected eAAV9 or AAV9 vectors encoding for SERCA2a in NAb+ post-myocardial infarction mice and further evaluated cardiac function using echocardiography. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at 6 weeks, respectively). In summary, delivery of AAVs protected by carrier exosomes (i.e. eAAVs) may retain the clinical benefits of AAVs while addressing one of its major challenges.

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Adeno-associated virus as a gene therapy vector: strategies to neutralize the neutralizing antibodies

Clinical and Experimental Medicine ◽

10.1007/s10238-019-00557-8 ◽

2019 ◽

Vol 19 (3) ◽

pp. 289-298 ◽

Cited By ~ 9

Author(s):

Majid Lotfinia ◽

Meghdad Abdollahpour-Alitappeh ◽

Behzad Hatami ◽

Mohammad Reza Zali ◽

Morteza Karimipoor

Keyword(s):

Gene Therapy ◽

Neutralizing Antibodies ◽

Adeno Associated Virus ◽

Gene Therapy Vector

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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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