rAAV Capsids Research Articles

Orthogonal characterization of rAAV9 reveals unexpected transgene heterogeneity

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10–40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of invivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

Rational Design of AAV-rh74, AAV3B, and AAV8 with Limited Liver Targeting.

Recombinant adeno-associated viruses (rAAVs) have become one of the leading gene therapies for treating a variety of diseases. One factor contributing to rAAVs' success is the fact that a wide variety of tissue types can be transduced by different serotypes. However, one commonality amongst most serotypes is the high propensity for liver transduction when rAAVs are administered peripherally. One of the few exceptions is the naturally occurring clade F AAV hematopoietic stem cell 16 (AAVHSC16). AAVHSC16 represents an interesting capsid in that it shows minimal liver transduction when injected peripherally. For capsids other than AAVHSC16, targeting non-liver tissues via peripheral AAV injection represents a challenge due to the high liver transduction. Thus, there is a demand for liver-de-targeted rAAV vectors. The rational design of rAAV capsids relies on current knowledge to design improved capsids and represents one means of developing capsids with reduced liver transduction. Here, we utilized data from the AAVHSC16 capsid to rationally design four non-clade F rAAV capsids that result in reduced liver transduction following peripheral injection.

Partial genome content within rAAVs impacts performance in a cell assay-dependent manner

Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance.

Application of Size Exclusion Chromatography with Multiangle Light Scattering in the Analytical Development of a Preclinical Stage Gene Therapy Program.

To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are required. However, these processes may introduce impurities that impact the performance and quality of the final drug product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical methods be implemented early in product development to measure the level of empty capsids. A process confirmation vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SV-AUC), droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at 260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content. This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce performance.

Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode.

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.

Enrichment of full rAAV capsids in a scalable, reproducible viral vector manufacturing platform

Recombinant adeno-associated viruses (rAAV) are the gene transfer vector of choice for many <i>in vivo</i> gene therapies. These vectors are synthetic viral particles which can deliver a therapeutic gene to a patient or patients’ cells to correct a genetic abnormality. These viral vectors can be produced in single-use bioreactors and purified using scalable single-use technologies. We evaluated the use of scalable, single-use filtration and chromatography technologies for downstream purification of an rAAV5 viral vector. In this testing, vector was produced in the Pall iCELLis^® Nano bioreactor by polyethylenimine (PEI) mediated triple-plasmid transfection. The harvest material was clarified using direct flow filtration with a combination of Seitz-P grade depth and 0.2 mm sterilizing grade filters. The product was concentrated using 100 kDa Omega^TM Membrane flat-sheet tangential flow-filtration (TFF) before primary purification with affinity chromatography. Affinity purified vector was polished using Mustang^® Q membrane chromatography to enrich for full capsids. The rAAV5 product was then concentrated and diafiltered to the final formulation using 100 kDa Omega TFF membrane. The final product was sterile filtered using Pall’s Supor^® EKV validated sterilizing-grade filters. This manufacturing process was optimized and evaluated for vector yield, low contaminant profile and full capsid enrichment. We established feasibility of a near complete end-to-end manufacturing process using almost all materials available from Pall Corporation. This process resulted in a theoretical whole process yield of ~25% with a low contaminant profile (host cell protein [HCP] and [DNA]) and a ~5-fold enrichment of full capsids to total capsids. The purification process described here shows potential for a scalable, platformable process for rAAV products.

EXTH-51. DEVELOPMENT OF A NOVEL GENE THERAPY APPROACH TARGETING GLIOBLASTOMA FOLLOWING ARTIFICIAL INTELLIGENCE (AI)-DIRECTED IDENTIFICATION OF THE GBM STATE

Abstract BACKGROUND Profound heterogeneity has severely hampered therapeutic advancements in GBM. Remarkably, however, GBM exhibits broad clinical and histopathologic overlaps suggesting the presence of a common state. The GBM state embodies network restructuring forced by founding mutations and perpetuated in subclones of GBM stem-like cells (GSCs). Successful targeting of the GBM state promises to circumvent the heterogeneity. METHODS To decipher the GBM state, we applied NETZEN, an AI suite integrating a deep neural network with gene network-based ranking, to first generate the reference GBM gene network from TCGA’s entire GBM RNAseq collection, and then identify the altered master regulatory gene subnetwork in GBM using a dataset containing &amp;gt;30 diverse patient-derived GSC lines and their paired differentiated cells, 6 astrocyte and 3 neuronal precursor cell lines. To develop a gene therapy against the GBM state, we screened a rAAV capsid library through GBM patient-derived xenografts (PDX) to identify variants with specific tropism for GBM cells that can deliver targeting constructs intratumorally. RESULTS We discovered a putative GBM state anchored by developmentally restricted master regulators. To validate its critical role, we deconstructed it using shRNA in a panel of PDX and uniformly observed improved tumor control and survival compared to controls (p&amp;lt; 0.05 in all lines). More notably, when the core GBM state was forcibly reconstructed in astrocytes, transformation into GSC-like cells occurred, as measured by single-cell analysis, neurosphere formation, and most importantly, development of lethal infiltrating brain tumors in 15/15 mice. Finally, selected novel rAAV capsids with 10-40-fold higher specificity for GBM cells were utilized in a shRNA-based rAAV platform to target key master regulators of the validated GBM state. CONCLUSIONS The GBM state is established by a developmental master regulator subnetwork permitting the creation of a first-of-its-kind, heterogeneity-agnostic GBM therapy. This AI-directed R&amp;D program can be expanded to target other tumors.

Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05–0.0001), in various mouse tissues in vivo (p < 0.03–0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing.

Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.

Enrichment of Fully Packaged Virions in Column-Purified Recombinant Adeno-Associated Virus (rAAV) Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography.

This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.

A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products

Recombinant adeno-associated virus (rAAV) is a vector with increasing popularity in the field of gene therapy. Like other drug substances manufactured in cell lines, rAAV vectors are commonly contaminated with host cell DNA, and the levels must be carefully monitored. The current method for residual DNA quantification in rAAV was adapted from protein programs and required sample digestion by proteinase prior to qPCR analysis. While the method worked effectively, it was unclear if proteinase digestion was essential for releasing DNA from rAAV capsids and improving qPCR efficiency. In this study, we systematically investigated the role of each component and treatment with the goal to simplify and streamline the method. It was determined that the proteinase digestion step was dispensable, while the addition of Tween 20 to rAAV samples was essential for accurate quantification of residual DNA. Based on this finding, a digestion-free method has been established that requires only a one-step sample preparation—addition of Tween 20. The method has been tested extensively with an rAAV9-based drug substance and process intermediates and verified with other rAAV serotypes. This significantly simplified and faster assay can be easily automated for high-throughput applications.

Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes.

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

Generation of Novel Cardiac Specific AAV Vectors by Directed Evolution in Human iPSC Derived Cardiomyocytes

Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the most promising gene therapy vectors. However, recent evidence has indicated that successful rAAV-mediated gene therapy in animal models may not translate to the same therapeutic benefit in humans. This is mainly due to the species difference in rAAV transduction efficiency, thus leading to the search for new capsids which exhibit high efficiency in human cells. This may be achieved through capsid shuffling, in which rAAV capsid gene sequences can be reconstituted from fragments of naturally occurring rAAV capsids to generate a library comprised of novel rAAVs with unique capsid protein sequences. This library could be screened using Directed Evolution (DE) in human cardiomyoctyes to select for cardiotropic rAAVs. In the development of strategies for personalised medicine, patient-specific iPSC-CMs could be used to evolve patent specific rAAVs. To test this strategy, we have used an rAAV library (rAAV1-12) to perform DE in human iPSC-CMs. The resulting rAAVs were then recovered from cell lysates and used for subsequent rounds of selection in fresh iPSC-CMs. After six rounds of selection, the recovered rAAVs were analysed for enrichment of cardiotropic candidates. Five candidates emerged. Functional analysis using rAAV-GFP indicated that while rAAV6 was the most efficient at transducing iPSC-CM among natural rAAVs, two of our novel variants led to higher GFP expression at the same vector dose. Our results validate the use of DE to develop novel, highly functional rAAVs for use in clinical studies.

Origins of truncated supplementary capsid proteins in rAAV8 vectors produced with the baculovirus system.

The ability to produce large quantities of recombinant Adeno-Associated Virus (rAAV) vectors is an important factor for the development of gene therapy-based medicine. The baculovirus/insect cell expression system is one of the major systems for large scale rAAV production. So far, most technological developments concerned the optimization of the AAV rep and cap genes in order to be expressed correctly in a heterologous system. However, the effect of the baculovirus infection on the production of rAAV has not been examined in detail. In this study we show that the baculoviral cathepsin (v-CATH) protease is active on several (but not all) rAAV serotypes, leading to a partial degradation of VP1/VP2 proteins. Subsequently, we identified the principal v-CATH cleavage site in the rAAV8 capsid proteins and demonstrated that the cleavage is highly specific. The proteolytic degradation of VP1/VP2 AAV capsid proteins reduces the infectivity of rAAV vectors but can be prevented by the use of a baculovirus vector with a deletion of the chiA/v-cath locus or by addition of the E64 protease inhibitor during production. Moreover, the codon optimization of AAV cap performed for several serotypes and originally aimed at the removal of potential alternative initiation codons, resulted in incorporation of additional forms of truncated VP1 into the rAAV capsids.

539. Screening for Recombinant Adeno-Associated Viral Vectors That Selectively Transduce Hepatitis B Virus Infected Cells

Hepatitis B is a disease caused by infection with the hepatitis B virus (HBV), a small enveloped DNA virus which belongs to the Hepadnaviridae family. Even though a safe and effective vaccine is available, HBV represents the ninth most common cause of death worldwide with an estimated 350 million chronically infected individuals. HBV infection can cause cirrhosis and hepatocellular carcinoma and currently there are 7 approved drugs for the treatment of HBV infection in the USA. These drugs decrease the risk of liver damage from HBV by slowing but not eliminating viral replication in most patients, possibly related to the persistence of integrated HBV genomes. Gene therapy using recombinant adeno-associated viral (rAAV) vectors constitute a promising tool to combat HBV infection as rAAV vectors have the ability to transduce and establish long-term and stable transgene expression in liver cells. Preclinical studies have successfully utilized rAAV vectors to combat viral infections after delivering short-hairpin RNA (shRNA) expression cassettes for knocking down viral coding or host genes required for viral replication and spread. Because promiscuous targeting of host cellular genes in the majority of non-infected cells may be detrimental, specific targeting of HBV-infected cells adds a new layer of potential therapeutic targets and safety. Thus we set out to identify new AAV capsids that selectively transduce HBV-infected cells. To do this, we created new AAV shuffled capsid libraries and used these for multiple rounds of infection in HBV-infected hepatoma cells. Each library is co-infected with wild-type Adenovirus-5 (helper virus) allowing the rAAV genomes to replicate in the HBV-infected cells. Isolation of replicating rAAV over successive passages in cultured cells allows for a more stringent selection as all steps in AAV transduction must occur. We have varied the screens using various multiplicity of infections (MOIs) of the library and/or utilized a pre-clearing step using a HBV negative hepatoma cell line. Several selected capsids derived from multiple parental capsids were vectorized. One capsid (AAV-GK4) was found to transduce the infected cells approximately 6 times more robustly than uninfected cells (using an MOI of 100 and 1,000). Interestingly, the same result was observed with AAV-DJ, a variant previously selected on human hepatoma cells. Additional screens and candidates are pending further evaluation. The possibility of finding new rAAV capsids with an increased transduction efficiency and selective tropism for HBV-infected cells will provide a reagent or therapeutic that can deliver a transgene that will allow one to selectively modify or potentially kill infected but not the uninfected cells contained within the liver.

305. A Screening Strategy for Selecting Recombinant Adeno-Associated Viral Vectors That Selectively Transduce Viral Infected Cells

Recombinant adeno-associated viral (rAAV) vectors constitute one of the most promising tools for gene transfer. Desired properties include the lack of pathogenicity in humans, broad tissue tropism, ability to transduce both dividing and non-dividing cells, and the potential to establish long-term and stable transgene expression in quiescent tissues. Additionally, the majority of transduction events are episomal, making the risk of insertional mutagenesis lower than integrating vectors. Preclinical studies have successfully utilized rAAV vectors to combat viral infections after delivering short-hairpin RNA (shRNA) expression cassettes for knocking down viral coding genes in infected cells. A viral infection can also be reduced using shRNAs against host cellular genes, which are required for the life cycle of the pathogen. Although combining both the targeting of viral and cellular genes has the potential to increase the success in combating viral infections, promiscuous targeting of host cellular genes in the majority of cells that remain uninfected in a tissue may be detrimental. Therefore, we have designed a strategy to select for rAAV capsids that will selectively transduce infected over uninfected cells. The selection is based on the fact that most viral infections cause phenotypic changes in a cell that likely affect at least one of the many parameters involved in rAAV transduction. For proof-of-concept, we are selecting for rAAV capsids that selectively transduce Hepatitis B virus (HBV)-infected hepatoma cells, in part because it was recently reported that HBV infection increases AAV2 and AAV8 transduction. We are currently characterizing a new highly diverse rAAV capsid library generated by shuffling of 18 parental capsids placed into a wild-type AAV genome. This library will be co-infected with wild-type Adenovirus-5 (helper virus) allowing the rAAV genomes to replicate in HBV-infected human cells. Isolation of replicating rAAV over successive passages in cultured cells will allow for stringent selection as all steps in AAV transduction, including cell entry, intracellular transport, nuclear entry and capsid uncoating, must occur in the selection scheme. After selection is completed, we will sequence and vectorize the most abundant capsids. These will be individually tested for their relative transduction of hepatoma cells that are identical except for their HBV infection status. Identifying such a rAAV capsid specific for HBV-infected cells will provide a reagent or therapeutic that can deliver a transgene that will allow one to selectively modify or potentially kill infected but not the uninfected cells contained within the liver. Moreover, we believe this strategy has the potential to be broadly applied for the selection of rAAV capsids specific for cells infected with other viruses or any disease state that results in phenotypic changes to a cell.

AAV-mediated gene therapy for metabolic diseases: dosage and reapplication studies in the molybdenum cofactor deficiency model

In a mouse model for molybdenum cofactor deficiency as an example for an inherited metabolic disease we have determined the dosage of recombinant AAV necessary to rescue the lethal deficiency phenotype. We demonstrated long-term expression of different expression cassettes delivered in a chimeric AAV capsid of serotype 1/2 and compared different routes of application. We then studied the effect of double and triple injections at different time points after birth and found a short neonatal window for non-response of the immune system. Exposition with rAAV capsids within this window allows transgene expression after a second rAAV transduction later. However, exposition within this window does not trigger immunotolerance to the viral capsid, which limits rAAV-mediated refurbishment of the transgene to only one more application outside this permissive window.

511. An Enzyme Linked Immunosorbent Assay for the Detection of Herpes Simplex Virus Type 1 Proteins in a Novel rAAV Production and Purification Process

AGTC has developed a recombinant herpes simplex virus (rHSV-1)-based adeno-associated virus (AAV) production process. This method requires two rHSV-1 viruses; rHSV-1-rep/cap which expresses the AAV2 rep and cap proteins, and a second rHSV-1 that contains the therapeutic gene of interest flanked by AAV2 inverted terminal repeats (ITRs) necessary for rAAV production. Following co-infection of these two helper viruses onto HEK-293 cells, the rAAV DNA is replicated and packaged into rAAV capsids. rAAV is then liberated from the cells and purified. Our proprietary rAAV purification scheme removes all contaminants including HSV-1 proteins; however, it is necessary to develop a sensitive and quantitative method to confirm the absence of HSV-1 proteins within a level of detection, in the final rAAV preparation.

Custom Adeno-Associated Virus Capsids: The Next Generation of Recombinant Vectors with Novel Tropism

Recombinant gene delivery vehicles based on adeno-associated virus (rAAV) have emerged as promising vectors for the correction of genetic and acquired human disease states. These vectors possess many characteristics, including low pathogenicity and immunogenicity, and long-term gene expression after a single administered dose, that make them leading candidates for clinical gene therapy applications. Yet, the broad tissue tropism of the available AAV serotypes remains a disadvantage for the safest, most effective in vivo delivery of transgenes to target tissues. In addition, clinically relevant cell types exist that are poorly transduced by current rAAV vectors. As a result, increased efforts are now being made to tailor the tropism of rAAV to improve their transduction and selectivity profiles. Flexible, diverse methodologies have emerged that allow more control over the cell surface receptors rAAV employs for cell entry. These novel rAAV production strategies have resulted in unique vectors characterized by unique capsid protein sequences that employ alternative receptors, and have provided a better understanding of many basic aspects of the rAAV life cycle. This review aims to summarize the genetic methods currently being employed to customize rAAV capsids.