rAAV Capsid Research Articles

A single dose recombinant AAV based CHIKV vaccine elicits robust and durable protective antibody responses in mice.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is responsible for Chikungunya fever, which is characterized by fever, rash, and debilitating polyarthralgia. Since its re-emergence in 2004, CHIKV has continued to spread to new regions and become a severe health threat to global public. Development of safe and single dose vaccines that provide durable protection is desirable to control the spread of virus. The recombinant adeno-associated virus (rAAV) vectors represent promising vaccine platform to provide prolonged protection with a single-dose immunization. In this study, we developed a rAAV capsid serotype 1 vector based CHIKV vaccine and evaluated its protection effect against CHIKV challenge. The recombinant AAV1 encoding the full-length structural proteins of CHIKV (named as rAAV1-CHIKV-SP) was generated in vitro by transfecting the plasmids of AAV helper-free system into HEK-293T cells. The safety and immunogenicity of rAAV1-CHIKV-SP were tested in 4-week-old C57BL/6 mice. The antibody responses of the mice receiving prime-boost or single-dose immunization of the vaccine were determined by ELISA and plaque reduction neutralizing test. The immunized mice were challenged with CHIKV to evaluate the protection effect of the vaccine. The rAAV1-CHIKV-SP showed remarkable safety and immunogenicity in C57BL/6 mice. A single dose intramuscular injection of rAAV1-CHIKV-SP elicited high level and long-lasting antibody responses, and conferred complete protection against a heterologous CHIKV strain challenge. These results suggest rAAV1-CHIKV-SP represents a promising vaccine candidate against different CHIKV clades with a simplified immunization strategy.

Assessment of adeno-associated virus purity by capillary electrophoresis-based western

A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Development of CNS tropic AAV1-like variants with reduced liver-targeting following systemic administration in mice

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes invivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

A novel method for quantitation of AAV genome integrity using duplex digital PCR

Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models.

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.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.

Direct ITR-to-ITR Nanopore Sequencing of AAV Vector Genomes.

Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.

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 >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< 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&D program can be expanded to target other tumors.

Vectored Immunotherapeutics for Infectious Diseases: Can rAAVs Be The Game Changers for Fighting Transmissible Pathogens?

Conventional vaccinations and immunotherapies have encountered major roadblocks in preventing infectious diseases like HIV, influenza, and malaria. These challenges are due to the high genomic variation and immunomodulatory mechanisms inherent to these diseases. Passive transfer of broadly neutralizing antibodies may offer partial protection, but these treatments require repeated dosing. Some recombinant viral vectors, such as those based on lentiviruses and adeno-associated viruses (AAVs), can confer long-term transgene expression in the host after a single dose. Particularly, recombinant (r)AAVs have emerged as favorable vectors, given their high in vivo transduction efficiency, proven clinical efficacy, and low immunogenicity profiles. Hence, rAAVs are being explored to deliver recombinant antibodies to confer immunity against infections or to diminish the severity of disease. When used as a vaccination vector for the delivery of antigens, rAAVs enable de novo synthesis of foreign proteins with the conformation and topology that resemble those of natural pathogens. However, technical hurdles like pre-existing immunity to the rAAV capsid and production of anti-drug antibodies can reduce the efficacy of rAAV-vectored immunotherapies. This review summarizes rAAV-based prophylactic and therapeutic strategies developed against infectious diseases that are currently being tested in pre-clinical and clinical studies. Technical challenges and potential solutions will also be discussed.

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques.

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities.

Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.

STEM-18. DEVELOPMENT OF A NOVEL GENE THERAPY APPROACH TARGETING GLIOBLASTOMA FOLLOWING AI-DIRECTED IDENTIFICATION OF A MASTER REGULATORY GENE NETWORK

Abstract BACKGROUND Profound heterogeneity has severely hampered therapeutic advancements in glioblastoma (GBM). Remarkably, GBM exhibits broad clinical and histopathologic overlaps suggesting the presence of a common regulatory state. The GBM state embodies restructuring of the master regulatory gene network (MRGN) forced by founding mutations and perpetuated in subclones of GBM stem-like cells (GSC). Successful targeting and altering of the MRGN promise to circumvent the heterogeneity. METHODS To decipher the common MRGN in GSC, we applied a robust AI suite, NETZEN, that integrates deep neural networks with gene network-based ranking to the reference GBM network generated using TCGA’s entire GBM RNAseq dataset and a dataset containing > 30 diverse GSC and paired GBM differentiated cell lines, 6 normal astrocyte and 3 normal neuronal precursor cell lines. To develop a gene therapy against the MRGN, we performed a screen of a rAAV capsid library using GBM patient-derived orthotopic xenografts (PDX) to identify capsid variants with specific tropism for GBM cells to deliver shRNA-based cassettes. RESULTS We discovered a putative MRGN in GSC, anchored by developmentally restricted master regulators (MR). To confirm its critical role, we deconstructed it using shRNA in a panel of PDX and uniformly observed attenuated tumor growth and mortality compared to controls (p < 0.05 in all lines). More notably, when normal astrocytes were forced to reconstruct the MRGN, transformation into GSC-like cells occurred, as measured by single-cell analysis, sphere formation in vitro, and most importantly, development of lethal infiltrating brain tumors in 15/15 mice. Finally, selected novel AAV capsids with 10-40-fold higher specificity for GBM cells were utilized in a shRNA-based AAV platform to target key MR in the validated MRGN. CONCLUSIONS The GBM state is established by a developmental subnetwork permitting a first-of-its-kind, heterogeneity-agnostic AAV-based treatment approach. This AI-directed R&D program can be expanded to target other cancers.

Bound Protein- and Peptide-Based Strategies for Adeno-Associated Virus Vector-Mediated Gene Therapy: Where Do We Stand Now?

Recombinant adeno-associated virus (rAAV) vectors have become one of the most promising and efficacious delivery vehicles for human gene therapy; however, low infectivity remains a major ongoing obstacle in the clinical application of rAAV vectors. Multiple strategies, including rAAV capsid modification and the application of pharmacological reagents, have been explored to enhance rAAV vector gene delivery. Recently, a new strategy using native proteins or various peptides has shown promise for increasing rAAV transduction locally or globally. This review summarizes the current status of protein- and peptide-based strategies and mechanisms to modulate rAAV transduction. We also provide a potential insight regarding the design of effective approaches for rAAV transduction enhancement in future clinical studies.

Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol

Endotoxin is the most common contaminant found in protein samples. Even a small amount of endotoxin can induce strong allergic reaction and death of a host organism. Endotoxin is also often detected in recombinant adeno-associated virus (rAAV) stocks prepared in research laboratories using off-the-shelf reagents; purifying rAAV stocks from endotoxin using commercial reagents sometimes results in significant titer loss. The problem is exacerbated due to the recently expanded diversity of rAAV serotypes and capsid variants, which, due to their variable capsid surface charge, display differential affinity toward endotoxin. In this paper, we describe a simple universal protocol of purifying vector stocks irrespective of AAV serotype. The protocol is based on subjecting endotoxin-contaminated rAAV to mild detergent treatment, followed by repeated buffer-exchange washing and concentrating viral stock by low-speed centrifugation. Multiple assays were employed to test the physical and biological equivalency of the viral stocks before and after purification. The described protocol has been routinely utilized to purify vector stocks contaminated at levels as high as >1,000 endotoxin units (EU)/mL to produce viral vectors with practically undetectable levels of endotoxin (<2.5 EU/mL), with the titer’s recovery in the range of 50%–100%.

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.

Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors.

PurposeEffective intravitreal gene delivery to cells of the central retina (i.e., photoreceptors) would be of substantial benefit for treating patients with retinal diseases, such as achromatopsia, where retinal detachment from a subretinal may be harmful. Previous studies demonstrated that mutation of the recombinant adeno-associated virus (rAAV) capsid through introduction of peptide insertions or amino acid substitutions dramatically alters vector tropism. Herein, we evaluate the photoreceptor transduction efficiency of three rAAV2/2-based capsid mutant vectors: rAAV2/2[7m8], rAAV2/2[QuadYF+TV], and a chimeric vector incorporating both mutations (termed rAAV2/2[MAX]) following intravitreal delivery in mice. Furthermore, we evaluate the transduction efficiency of rAAV2/2[MAX] using explanted human central retinal samples to address clinical translatability.MethodsVectors containing a GFP or mCherry reporter gene were intravitreally injected into C57BL/6J or Nrl-EGFP mice, respectively. Transduction was assessed in vivo utilizing a custom multiline confocal scanning laser ophthalmoscope. Injected Nrl-EGFP mouse retinas were used to quantify transduced photoreceptors using flow cytometry. Postmortem human retinal tissue was cultured following administration of rAAV2/2[MAX]. C57BL/6J retinas and human explants were cryosectioned to determine vector tropism.ResultsThe chimeric vector rAAV2/2[MAX] transduced significantly higher proportions of the retina than did either single mutant serotypes following intravitreal delivery in murine retina, including inner retinal cells and photoreceptors. Vector rAAV2[MAX] demonstrated transduction of human photoreceptors and ganglion cells.ConclusionsTransduction observed via rAAV2/2[MAX] indicates that combining mutations with complementary mechanisms of action in a single vector results in enhanced transduction. rAAV2/2[MAX] also presented the ability to transduce human photoreceptors and ganglion cells, indicating potential for efficient intravitreal vector delivery.