Serotype-specific replicating AAV helper constructs increase recombinant AAV type 2 vector production

One of the major limitations of the use of adeno-associated virus (AAV) as a tool for gene therapy is the difficulty in providing sufficient quantities of the virus for pre-clinical and clinical trials. Here, we report a novel approach for amplifying AAV helper functions, which mimics the normal replication of wild type (wt) AAV resulting in a high yield of AAV vectors. Cotransfection of replicating but non-packaging AAV helper constructs in the presence of adenovirus (Ad) produces a high level of Rep and Cap proteins. Yield of AAV2/GFP vector obtained from this helper DNA replication system was approximately 20-fold higher than traditional methods. Molecular analysis suggested that virus yield was associated with capsid protein production. The transfection ratio was optimized using these novel helper constructs, resulting in an additional 2-fold increase in vector yield without presence of replication competent AAV (rcAAV). This strategy supports development of AAV packaging systems that retain normal virus replication capability without helper virus encapsidation

Building a Better Vector: The Manipulation of AAV Virions

This review will focus on research directed at manipulating the virion of adeno-associated virus (AAV) with the goals of circumventing the immune response of the virion, as well as retargeting the virus to specific cell types of interest. The use of five AAV serotypes for addressing questions of Ab neutralization, novel tropism, as well as providing natural templates for targeting by virion modification will be discussed

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system

Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection

Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective

Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications

An Emerging Adeno-Associated Viral Vector Pipeline for Cardiac Gene Therapy

The naturally occurring adeno-associated virus (AAV) isolates display diverse tissue tropisms in different hosts. Robust cardiac transduction in particular has been reported for certain AAV strains. Successful applications of these AAV strains in preclinical and clinical settings with a focus on treating cardiovascular disease continue to be reported. At the same time, these studies have highlighted challenges such as cross-species variability in AAV tropism, transduction efficiency, and immunity. Continued progress in our understanding of AAV capsid structure and biology has provided the rationale for designing improved vectors that can possibly address these concerns. The current report provides an overview of cardiotropic AAV, existing gaps in our knowledge, and newly engineered AAV strains that are viable candidates for the cardiac gene therapy clinic

Recombinant adeno-associated virus vectors in the treatment of rare diseases

An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use

Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex

ABSTRACT Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae . The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. IMPORTANCE Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting helper virus, such as adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications for understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance and likely translate to other viruses that must navigate the DDR

Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction