Adeno-associated Virus Libraries Research Articles

AAV library screening identifies novel vector for efficient transduction of human aorta.

Targeted gene delivery to vascular smooth muscle cells (VSMCs) could prevent or improve a variety of diseases affecting the vasculature and particularly the aorta. Thus, we aimed to develop a delivery vector that efficiently targets VSMCs. We selected engineered adeno-associated virus (AAV) capsids from a random AAV capsid library and tested the top enriched motifs in parallel screening through individual barcoding. This approach allowed us to distinguish capsids that only transduce cells based on genomic DNA (gDNA) from those also mediating transgene expression based on transcribed cDNA reads. After three rounds of selection on primary murine VSMCs (mVSMCs), we identified a novel targeting motif (RFTEKPA) that significantly improved transduction and gene expression efficiency over AAV9-wild type(WT)and increased expression in mVSMCsby70% compared to thepreviously identified SLRSPPS peptide. Further analysis showed that the novel motif also improved expression in human aortic smooth muscle cells (HAoSMCs) and human aortic tissue ex vivo up to threefold compared to SLRSPPS and approximately 70-fold to AAV9-WT. This high cross-species transduction efficiency makes the novel capsid motif a potential candidate for future clinical application in vascular diseases.

AAVolve: Concatenated long-read deep sequencing enables whole capsid tracking during shuffled AAV library selection

Gene therapies using recombinant adeno-associated virus (AAV) vectors have demonstrated considerable clinical success in the treatment of genetic disorders. Improved vectors with favourable tropism profiles, decreased immunogenicity and enhanced manufacturability are poised to further improve the state of gene therapies. Such vectors can be identified through directed evolution, a process of subjecting a diverse capsid library to a selection pressure to identify individual variants with a desired trait. Currently, libraries that involve changes distributed throughout the AAV capsid coding region, such as DNA family shuffled libraries, are largely characterised using low-throughput Sanger sequencing of individual clones. However, improvements in long-read sequencing technologies have increased their applicability to capsid libraries and evaluation of the selection process. Here we explore the application of Oxford Nanopore Technologies refined by a concatemeric consensus method for initial library characterization and monitoring selection of a shuffled AAV capsid library. Furthermore, we present AAVolve, a bioinformatic pipeline for processing long-read data from AAV directed evolution experiments. Our approach allows high throughput characterisation of AAV capsids in a streamlined manner, facilitating deeper insights into library composition through multiple rounds of selection, and generalization through training of machine learning models.

<i>In vivo</i> evaluation of tropism and biodistribution of synthetic and natural adeno-associated viral vectors by next-generation sequencing

INTRODUCTION. The creation of synthetic adeno-associated virus (AAV) vectors during gene therapy development is a labour-intensive and expensive process. The optimal solution to minimise the time and costs associated with gene therapy development lies in the improvement of methods aimed at assessing AAV vector biodistribution and transduction efficiency in vivo.AIM. This study aimed to develop a new bioinformatics-based assessment method for synthetic AAV vector libraries to analyse AAV vector biodistribution and transduction efficiency in vivo.MATERIALS AND METHODS. The production of synthetic AAV vectors involved assigning AAV serotype-specific barcodes (12-nucleotide tags flanked at the 5' end with a sequence encoding the green fluorescent reporter protein). Plasmids carrying unique barcodes were propagated in competent Escherichia coli XL10-Gold cells and used to create two AAV libraries: L1 with a viral genome count of 1010 and L2 with a viral genome count of 1011. AAV production involved HEK293T cell transfection. L1 and L2 library vectors were administered to C57Bl/6N mice by intravenous injection. DNA and RNA were isolated from transduced organs for analysis by next-generation sequencing. The obtained data on DNA and RNA barcode quantities in different murine organs were analysed to assess the biodistribution and transduction efficiency of synthetic AAVs. Barcodes were identified by aligning them to the expected sequences and counted. The resulting values were normalised to the quantity of barcodes in the original library.RESULTS. Seven viral constructs based on different AAV serotypes were created as part of two AAV libraries. Six of the AAV serotypes were synthetic (sAAV1, sAAV2, sAAV3, sAAV4, sAAV5, and sAAV6). Sequencing of murine organ samples revealed significant quantities of DNA barcodes from both AAV libraries in all organs except the brain. For the L1 library, RNA barcodes were detected at a sufficient level in 4 organs, including the skeletal muscles, the heart, the liver, and the adrenal glands. For the L2 library, in addition to the listed organs, sufficient RNA-barcode levels were observed in the gonads and the kidneys. According to transduction efficiency analysis based on RNA barcode levels adjusted for DNA barcodes, sAAV5 was considered the most promising variant for gene therapy of liver-related diseases, whereas sAAV2 and sAAV6 were recognised as holding the most promise for adrenal diseases.CONCLUSIONS. The developed bioinformatics-based assessment method for synthetic AAV vector libraries can analyse AAV vector biodistribution and transduction efficiency in the body. The presented approach has the potential for selecting optimal AAV vectors for specific organs and tissues in further gene therapy development.

Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery.

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

THER-01. Precision brain tumor therapy by AAV-mediated oncogene editing

Pediatric high-grade glioma is a heterogeneous group of highly malignant tumors of the central nervous system, with a median overall survival of less than two years after diagnosis, demanding novel treatment options. One innovative approach is gene therapy, which has so far been hampered for cancer treatment owing to the lack of a system targeting tumor cells specifically. To overcome this limitation, we established a novel strategy for gene therapy, combining tumor cell-specific adeno-associated virus (AAV) variants with oncogene-specific CRISPR-Cas nucleases. We screened 177 different Cas9/gRNA combinations targeting the genes encoding H3K27M or BRAFV600E, and identified highly specific nucleases that edited the oncogenic allele but left the respective WT loci intact, which we validated by PCR amplicon sequencing. Next, we intravenously injected an AAV library engineered to encode its own capsid DNA into mice harboring patient-derived xenograft tumors driven by H3K27M or BRAFV600E. After 21 days, we resected neoplasms and separated mCherry-labeled tumor cells from normal surrounding cells by fluorescence-activated cell sorting. Using the DNA from tumor cells as template, we generated a second AAV library, which was utilized in another round of in vivo selection. At the end of each screen, DNA from tumor cells, surrounding cells, and control tissues (liver and spleen) was analyzed by amplicon sequencing. Strikingly, we identified multiple AAV variants that were highly and recurrently enriched in the analyzed tumor tissues. We are currently validating these variants by intravenously injecting selected, GFP-encoding AAVs to tumor-bearing mice and by subsequently analyzing their distribution throughout the aforementioned tissues. We will combine oncogene-specific nucleases with these validated AAV variants and analyze their anti-tumoral efficacy in a preclinical setting. Furthermore, we plan to adapt this approach to allografted mice, evaluating its feasibility and efficacy in syngeneic models.

A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain. Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.

Directed evolution of AAV accounting for long-term and enhanced transduction of cardiovascular endothelial cells in vivo

Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.

Frustration and Direct-Coupling Analyses to Predict Formation and Function of Adeno-Associated Virus

Adeno-associated virus (AAV) is a promising gene therapy vector because of its efficient gene delivery and relatively mild immunogenicity. To improve delivery target specificity, researchers use combinatorial and rational library design strategies to generate novel AAV capsid variants. These approaches frequently propose high proportions of nonforming or noninfective capsid protein sequences that reduce the effective depth of synthesized vector DNA libraries, thereby raising the discovery cost of novel vectors. We evaluated two computational techniques for their ability to estimate the impact of residue mutations on AAV capsid protein-protein interactions and thus predict changes in vector fitness, reasoning that these approaches might inform the design of functionally enriched AAV libraries and accelerate therapeutic candidate identification. The Frustratometer computes an energy function derived from the energy landscape theory of protein folding. Direct-coupling analysis (DCA) is a statistical framework that captures residue coevolution within proteins. We applied the Frustratometer to select candidate protein residues predicted to favor assembled or disassembled capsid states, then predicted mutation effects at these sites using the Frustratometer and DCA. Capsid mutants were experimentally assessed for changes in virus formation, stability, and transduction ability. The Frustratometer-based metric showed a counterintuitive correlation with viral stability, whereas a DCA-derived metric was highly correlated with virus transduction ability in the small population of residues studied. Our results suggest that coevolutionary models may be able to elucidate complex capsid residue-residue interaction networks essential for viral function, but further study is needed to understand the relationship between protein energy simulations and viral capsid metastability.

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.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex more than 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent and sustained in two mouse strains (C57BL/6 and BALB/c), making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F.

Impact of the Assembly-Activating Protein on Molecular Evolution of Synthetic Adeno-Associated Virus Capsids.

Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.

InVivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ.

Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent invivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

How to Successfully Screen Random Adeno-Associated Virus Display Peptide Libraries In Vivo.

Adeno-associated virus (AAV) has emerged as a very promising gene therapy vector. To enable tissue-directed gene expression, many artificially generated AAV variants have been established, often isolated from large pools of mutated capsids. Random peptide libraries displayed on AAV capsids have been used successfully to select vectors targeted to a given target cell or tissue in vitro and in vivo. However, the published methodology for screening of AAV libraries to isolate vectors with selective tissue tropism after intravenous administration in vivo has not been described in sufficient detail to address all critical steps. A step-by-step protocol is provided here.

542. Novel Barcode-Based In Vivo Screening Method for Generating De Novo AAV Serotypes for CNS-Directed Gene Therapy

Capsid modification is a useful strategy to create adeno-associated virus (AAV) vectors with subtype specific neuronal targeting and enhanced retrograde transport. Incorporating known cell-specific binding ligands is a rational method, but the creation of vectors without prior knowledge has the potential to reveal novel targets. Directed evolution and phage display are broadly utilized high-throughput methods, but are inefficient due to displaying random peptides wherein the vast majority will be non-functional.Here, we have developed a novel AAV library in which each virus particle display a peptide derived from known neuron-related proteins on the surface of an AAV2 capsid. The packaged viral genome encodes a unique barcode sequence to facilitate capsid identification. 92398 unique oligos encoding 14-amino-acid peptides derived from 135 proteins were synthesized using microarray. Four-fragment Gibson assembly and novel emulsion PCR was then used to generate a plasmid library by inserting oligos into the capsid gene and barcodes between the inverted terminal repeats. This plasmid library was then used to assemble a diverse library of AAV capsids, such that particles were composed of only peptide-modified capsid proteins which package an expression cassette containing RNA expressed barcodes providing post hoc identification of the capsid structure.In parallel, the plasmid library was sequences using Illumina paired-end sequencing to link the RNA expressed barcodes to the de novo capsid structures. The successfully generated AAV library efficiently infected neurons and astrocytes in vitro and displayed a subset of peptides that had efficient retrograde transport ability in neurons in vivo (e.g., transported from striatum to substantia nigra). Functional peptides, which successfully promoted neuronal infectivity or retrograde transport, were identified by Illumina sequencing of RNA expressed barcodes both in vitro and in vivo and efficacy modeled through barcode counting. In conclusion, we developed a high-throughput combinatorial method to generate peptide-modified AAV libraries that are valuable for evaluation of receptor expression of neuronal populations and have the potential to generate novel vectors with unique properties for in vivo gene transfer in the CNS.Figure 1Polar plot showing the number of unique peptides found in custom array and plasmid library followed by the number found in infective AAV particles, at injection site and for transported AAVs.View Large Image | Download PowerPoint Slide

257. Selection of Next Generation AAV Gene Therapy Vectors for Specific and Precise Gene Delivery

The ultimate goal in human gene therapy is the specific and exclusive modification of the desired target cells upon systemic vector administration. Especially vectors derived from adeno-associated virus (AAV) are among the most promising gene transfer systems for in vivo application and have received broad attention due to substantial clinical benefit. However, AAV specificity for a particular target cell or tissue has been hampered by the broad tropism of different AAV serotypes. Over the last several years, new approaches have been initiated to create and select for more effective and selective recombinant AAV vectors by genetically modifying the capsid protein. These methods include random and/or rationale amino acid substitutions, creation of chimeric capsid variant libraries and various selection screens, and/or peptide insertion. A different approach involves the incorporation of highly specific binding molecules (DARPins) into the AAV capsid. DARPins are derived from ankyrin-repeat proteins that have been developed as alternative to antibody-based scaffolds, which are selected by high-throughput screens from DARPin libraries. At present, the bottleneck remains the cumbersome selection of DARPin molecules and the low number of functional DARPin capsid chimeras, which are able to assemble into a functional vector and still bind their intended extracellular target. Thus after months of screening and analysis the majority of the pre-selected DARPins are found not to be suitable for AAV targeting approaches. Here we describe a novel reliable and faster selection approach. First we eliminate the cumbersome and often misleading prokaryotic selection steps. To do this, we incorporate an entire DARPin library into the AAV capsid as VP2-fusion protein, generating either replication deficient or competent AAV library systems allowing for direct screening on therapeutically relevant cell types in vitro or in vivo. Our studies show that replicating deficient and replicating AAV libraries can be generated with high diversity up to 5×107 and high functional titers retaining their infectivity. We have generated a DARPin-capsid library in AAV-DJ (serotype with diverse cellular tropism) and AAV-LK03 (human selective serotype) and after only two selection rounds, found up to 10% of selected DARPins sharing identical repeat motifs responsible for target receptor binding in the pancreas and the liver. We are currently making vectors from these chimeric capsids and will determine their target specificity both in vivo and in vitro. This new approach takes only six days per selection cycle, allowing discovery of novel DARPin molecules and selection for targeted vectors in substantially reduced time. This approach expands the potential diversity in creating and defining novel rAAV vectors with medically relevant transduction properties. Such viral vectors will not only open the door for an array of new approaches to treat acquired human diseases but also push the development of new AAV-based gene therapy vectors to the next level.

96. An Approach to Compare Multiple Adeno-Associated Virus (AAV) Capsids Side-by-Side in a Single Eye Following Intravitreal Injection

Current gene therapy approaches for ocular diseases rely largely on the use of subretinal injection for treatment of outer nuclear layer cells of interest. While subretinal injection allows for efficient transduction of retinal cells close to the site of the injection, there is a significant risk that the injection can further damage retinas with compromised integrity. Several studies have identified new adeno-associated virus (AAV) capsid mutants that are potentially able to transduce outer nuclear layer cells following intravitreal injections. To further explore AAV vector-mediated intravitreal injection approaches for clinical translation, it is imperative to directly compare retinal transduction efficiencies between newly identified and previously published AAV mutants. Using clinically relevant large animal models, these mutant capsids, as well as other commonly used AAV serotypes, can be tested in a comprehensive manner. To this end, we propose to utilize a viral DNA/RNA barcoding approach established by our lab. In this approach, AAV capsids derived from different serotypes and mutants can be identified by DNA and RNA barcodes unique to each AAV capsid. These barcodes are contained in viral genomes and also expressed as RNA barcodes following cellular transduction. As each AAV capsid strain has a unique viral genome, a mass transduction of a number of different AAV strains can be conducted in order to directly compare transduction levels side-by-side in a single eye. To test feasibility of the barcode approach and establish proof-of-principle in the ocular gene therapy research, we produced a DNA/RNA-barcoded AAV library that contained 20 serotypes including AAV serotypes 1-11 and AAV mutants including DJ, LK03, 2i8 and 2G9. Each AAV strain expresses a pair of 12 nucleotide-long RNA barcodes unique to each strain under the control of the human U6 snRNA promoter. Seven C57BL/6J mice have been injected into vitreous humor bilaterally with the DNA/RNA-barcoded AAV library vector at doses ranging from 2 to 9 x109 vg. To date, eyes were harvested from one mouse 3 weeks post injection. Total DNA and RNA were extracted from the retina as a whole from each eye, and were subjected to DNA-PCR or reverse transcriptase (RT)-PCR to amplify viral DNA and RNA barcodes, followed by Illumina barcode sequencing. A preliminary data analysis revealed that AAV2, AAV-DJ and AAV2G9 transduced the murine retina better than other AAV strains including AAV9, the reference control AAV strain contained in the library. Although further optimization of the approach may be required to move this project forward, we plan to apply this method to assess transduction efficiencies of various AAV strains side by side in specific subsets of sorted retinal cells and in various animal species. The safer approach of intravitreal injections with the most optimal AAV vectors would significantly increase the ability to treat over 200 genetic retinal diseases and therefore the data that will be obtained from these studies will considerably benefit the field of retinal gene therapy.

AAV ancestral reconstruction library enables selection of broadly infectious viral variants.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy.

262. Directed Evolution of Improved AAV Capsids for the Ideal Human Liver Vector–Can Human Liver Tropism and Human Immune Evasion Be Achieved?

Research towards improved adeno-associated virus (AAV) delivery vectors for use in treating human liver diseases continues to be dominated by efforts to characterize potential candidate vectors in mice which often can't recapitulate either the human liver tropism/transduction profiles, or the specifics of the human immune response to these vectors. Research from our lab has shown that despite the impressive hepatocyte transduction levels by AAV-8 in mouse liver, AAV-8 does not functionally transduce human hepatocytes in xenografted humanized liver mouse models to levels of therapeutic relevance, possibly explaining the lower than expected FIX expression levels from the recent AAV-8 hemophilia clinical trial. Efforts to evolve novel capsid variants with improved human hepatocyte tropism were successful with the generation of AAV-LK03, yet limitations to clinical translation with many newly evolved serotypes remains due to high levels of pre-existing neutralizing antibodies that may cross-react and neutralize transduction. For this reason we sought to evolve improved hepatotropic variants–again in humanized liver mice–that not only retained robust human liver tropism, but also had been evolved for resistance to pre-existing neutralizing antibodies in the human population to create the ideal liver vector for use in the clinic. We utilized wild-type replicating AAV libraries of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when co-administered with wild-type adenovirus, making chimeric humanized liver mice an excellent tool to allow for selection of capsids with tropism for human liver. Screens were carried out for five rounds of selection and rather than waiting for the screen to go to completion, we ended the screen with some library diversity remaining for use in a subsequent screen for neutralizing antibody evasion. All variants from round five of the human liver screen were carried forward and screened for two additional rounds for their ability to resist binding to pooled human immunoglobulins in IgG immunocapture assays. The top 100 highly selected variants were sequenced and vectorized into CAG-driven GFP preparations in seven pools based on capsid relatedness at the amino acid level. Each pool was then resubjected to additional pooled human immunoglobulin screening and the best pool was chosen. The top seven candidates from this pool were tested alongside control serotypes that represent the extremes for humoral neutralization: highest neutralization (AAV-2), lowest neutralization (AAV-DJ), as well as LK03 that falls in between. One variant from this highly selected pool has neutralization profiles on par with AAV-DJ and further characterizations are underway in humanized liver mice in vivo and various human hepatoma lines in vitro to determine if both desired characteristics were robustly retained—human liver tropism and human immune evasion.