Adeno-associated Virus Gene Delivery Research Articles

Optimization of an adeno-associated viral vector for epidermal keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

Gene therapy strategies and prospects for neurofibromatosis type 1

To summarize the gene therapy strategies for neurofibromatosis type 1 (NF1) and related research progress. The recent literature on gene therapy for NF1 at home and abroad was reviewed. The structure and function of the NF1 gene and its mutations were analyzed, and the current status as well as future prospects of the transgenic therapy and gene editing strategies were summarized. NF1 is an autosomal dominantly inherited tumor predisposition syndrome caused by mutations in the NF1 tumor suppressor gene, which impair the function of the neurofibromin and lead to the disease. It has complex clinical manifestations and is not yet curable. Gene therapy strategies for NF1 are still in the research and development stage. Existing studies on the transgenic therapy for NF1 have mainly focused on the construction and expression of the GTPase-activating protein-related domain in cells that lack of functional neurofibromin, confirming the feasibility of the transgenic therapy for NF1. Future research may focus on split adeno-associated virus (AAV) gene delivery, oversized AAV gene delivery, and the development of new vectors for targeted delivery of full-length NF1 cDNA. In addition, the gene editing tools of the new generation have great potential to treat monogenic genetic diseases such as NF1, but need to be further validated in terms of efficiency and safety. Gene therapy, including both the transgenic therapy and gene editing, is expected to become an important new therapeutic approach for NF1 patients.

Structural and Biophysical Analysis of Adeno-Associated Virus Serotype 2 Capsid Assembly Variants.

Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability (Tm) than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. IMPORTANCE Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.

Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Self-Complementary Adeno-Associated Viral Gene Delivery in the Metanephric Organ Culture Identifies Pvt1 as Modulator of Cystogenesis

Self-Complementary Adeno-Associated Viral Gene Delivery in the Metanephric Organ Culture Identifies Pvt1 as Modulator of Cystogenesis

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Enhancement of transduction efficiency using Adeno-associated viral vectors by chemical pretreatment to mice bladder urothelium

Adeno-associated virus (AAV) vectors have been recognized as promising tools for gene delivery. The bladder is a seemingly ideal organ for virus transfer, with easy access through the urethra enabling organ-specific delivery. However, achieving adequate transduction efficiency in the urothelium has been a major challenge because of the barrier function of the glycosaminoglycan (GAG) layer. We investigated optimal pretreatments of the bladder urothelium to maximize transduction efficiency by AAV vectors in vivo. Murine bladders were pretreated with five different chemical agents followed by transurethral instillation with an AAV2 vector encoding a tdTOMATO reporter. After 7 days, transduction efficiency of the urothelium was evaluated. Bladder urothelia pretreated with HCl showed clear evidence of AAV infection and gene delivery. Mice treated with 0.1 N HCl for 4 min showed significantly higher survival rates (nearly 80 %) compared with mice receiving other pretreatment regimens. AAV vector transduction in the urothelium was observed in seven of 20 mice (35 %), and the mean transduction efficiency in these mice was 14.5 %. Thus, HCl pretreatment enhanced transduction efficiency of the mice bladder urothelium by an AAV vector in vivo. Pretreatment with 0.1 N HCl for 4 min was the optimal condition to maximize survival and transduction efficiency of the urothelium.

APOE4 exacerbates α-synuclein pathology and related toxicity independent of amyloid.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease mainly by driving amyloid-β pathology. Recently, APOE4 has also been found to be a genetic risk factor for Lewy body dementia (LBD), which includes dementia with Lewy bodies and Parkinson's disease dementia. How APOE4 drives risk of LBD and whether it has a direct effect on α-synuclein pathology are not clear. Here, we generated a mouse model of synucleinopathy using an adeno-associated virus gene delivery of α-synuclein in human APOE-targeted replacement mice expressing APOE2, APOE3, or APOE4. We found that APOE4, but not APOE2 or APOE3, increased α-synuclein pathology, impaired behavioral performances, worsened neuronal and synaptic loss, and increased astrogliosis at 9 months of age. Transcriptomic profiling in APOE4-expressing α-synuclein mice highlighted altered lipid and energy metabolism and synapse-related pathways. We also observed an effect of APOE4 on α-synuclein pathology in human postmortem brains with LBD and minimal amyloid pathology. Our data demonstrate a pathogenic role of APOE4 in exacerbating α-synuclein pathology independent of amyloid, providing mechanistic insights into how APOE4 increases the risk of LBD.

A ligand-based system for receptor-specific delivery of proteins.

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems

Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.

Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Design of AAV Vectors for Delivery of Large or Multiple Transgenes.

Adeno-associated virus (AAV)-mediated gene therapy has evolved from bench to bedside, and now is the therapy of choice for certain inherited diseases. However, the small packaging capacity of AAV vectors prevents this technique from treating genetic diseases with mutations of large genes. Multiple strategies, including split AAV gene delivery and oversized AAV gene delivery, have been explored to deliver large gene expression cassettes. These strategies have gained some success in animal experiments. In this chapter, we review the progress of AAV-mediated delivery of large expression cassettes. We also review using AAV to deliver multiple transgenes.

APOE \u03b52 is associated with increased tau pathology in primary tauopathy

Apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease mainly by modulating amyloid-β pathology. APOE ε4 is also shown to exacerbate neurodegeneration and neuroinflammation in a tau transgenic mouse model. To further evaluate the association of APOE genotype with the presence and severity of tau pathology, we express human tau via an adeno-associated virus gene delivery approach in human APOE targeted replacement mice. We find increased hyperphosphorylated tau species, tau aggregates, and behavioral abnormalities in mice expressing APOE ε2/ε2. We also show that in humans, the APOE ε2 allele is associated with increased tau pathology in the brains of progressive supranuclear palsy (PSP) cases. Finally, we identify an association between the APOE ε2/ε2 genotype and risk of tauopathies using two series of pathologically-confirmed cases of PSP and corticobasal degeneration. Our data together suggest APOE ε2 status may influence the risk and progression of tauopathy.

Screening for Neutralizing Antibodies Against Natural and Engineered AAV Capsids in Nonhuman Primate Retinas.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.

Immune responses in gene therapy with adeno-associated virus (AAV) vectors have been the object of almost two decades of study. Although preclinical models helped to define and predict certain aspects of interactions between the vector and the host immune system, most of our current knowledge has come from clinical trials. These studies have allowed development of effective interventions for modulating immunotoxicities associated with vector administration, resulting in therapeutic advances. However, the road to full understanding and effective modulation of immune responses in gene therapy is still long; the determinants of the balance between tolerance and immunogenicity in AAV vector-mediated gene transfer are not fully understood, and effective solutions for overcoming preexisting neutralizing antibodies are still lacking. However, despite these challenges, the goal of reliably delivering effective gene-based treatments is now in sight.

Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models.

Currently, clinical gene therapy is experiencing a renaissance, with new products for clinical use approved in Europe and clinical trials for multiple diseases reporting positive results, especially those using recombinant adeno-associated viral (rAAV) vectors. Amid this new success, it is prudent to recall that the field of gene therapy experienced tragic setbacks in 1999 and 2002 because of the serious adverse events related to retroviral and adenoviral gene delivery in two clinical trials that resulted in the death of two patients. In both cases, the toxicity observed in humans had been documented to occur in animal models. However, these toxicities were either undetected or underappreciated before they arose in humans. rAAVs have been tested extensively in animals and animal models of disease, largely without adverse events, except for transient elevation in liver enzymes in some patients. However, a small but growing number of murine studies have documented that adeno-associated viral gene delivery can result in insertional mutagenesis. Herein, the aggregate data are reviewed from multiple murine studies where genotoxicity associated with rAAV treatment has been observed. The data emphasize the need for a proactive position to evaluate the potential risks and possible solutions associated with AAV-mediated gene therapy.

Induction of T-Cell Infiltration and Programmed Death Ligand 2 Expression by Adeno-Associated Virus in Rhesus Macaque Skeletal Muscle and Modulation by Prednisone

Use of adeno-associated virus (AAV) to transduce genes into skeletal muscles can be associated with T-cell responses to viral capsid and/or to transgenic protein. Intramuscular mononuclear cell infiltrates primarily consisting of CD8+ T cells and also containing FOXP3+ regulatory T cells were present in rhesus macaque skeletal muscle treated with rAAVrh74.MCK.GALGT2 by vascular delivery. Administration of oral prednisone prior to AAV gene delivery and throughout the study reduced such infiltrates by 60% at 24 weeks post AAV delivery compared with AAV-treated animals not receiving prednisone, regardless of the presence of pre-existing AAV serum antibodies at the time of treatment. The majority of CD8+ T cells in AAV-treated muscles expressed activated caspase 3 and programmed cell death protein 1 (PD1), suggesting ongoing programmed cell death. AAV-transduced skeletal muscles also had elevated expression of programmed death ligand 2 (PDL2) on skeletal myofibers, and this increase in expression extended to muscles where transgene was not overexpressed. These data demonstrate that prednisone can reduce the extent of intramuscular T-cell infiltrates in AAV-treated muscles, which may aid in achieving long-term transgene expression, as may the induction of PDL2 expression on skeletal myofibers to promote PD1-mediated programmed T-cell death.

Abstract 13093: An Imbalance in Cardiomyocyte Glucocorticoid and Mineralocorticoid Receptor Signaling Leads to Heart Failure

Stress is increasingly associated with adverse cardiac outcomes; however the mechanisms underlying these effects are poorly understood. Glucocorticoids are primary stress hormones that regulate cell and tissue homeostasis through two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Cardiomyocytes express both these receptors but little is known concerning their coordinated actions in heart physiology and whether an imbalance in their signaling contributes to cardiac disease. Here, we show that patients with failing hearts have a disparity in the relative levels of GR and MR compared to non-failing donor hearts. To examine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioMRKO mice exhibited normal heart function whereas the cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction. Surprisingly, the cardioGRMRdKO mice were protected from the cardiac disease observed for the cardioGRKO mice. At a molecular level, pathological gene changes present in the GR deficient hearts were also found in the double knockout hearts whereas specific cardioprotective gene changes were detected only in hearts from the cardioGRMRdKO mice. Re-installation of MR into the cardioGRMRdKO hearts by adeno-associated virus gene delivery reversed these cardioprotective gene changes and resulted in cardiac dysfunction. These findings reveal critical gene-regulatory roles for both GR and MR in the heart and suggest that an imbalance in these two signaling pathways leads to heart disease. Therapies designed to modulate cardiomyocyte glucocorticoid signaling to favor more GR and less MR activity may provide new approaches for treating the failing heart.

Identification and Validation of Small Molecules That Enhance Recombinant Adeno-associated Virus Transduction following High-Throughput Screens.

While the recent success of adeno-associated virus (AAV)-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2,396 compounds. We identified 13 compounds that were capable of enhancing transduction, of which 12 demonstrated vector-specific effects and 1 could also enhance vector-independent transgene expression. Many of these compounds had similar properties and could be categorized into five groups: epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors. In vivo analysis of rAAV2 and all of the clinically relevant compounds revealed that, consistent with our in vitro results, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectors in vivo using an AAV8 capsid that is known to exhibit robust liver tropism. Consistent with our in vitro results, teniposide coadministration greatly enhanced fragmented rAAV8 transduction at 48 h and 8 days. This study provides a foundation based on the rAAV small-molecule screen methodology, which is ideally used for more-diverse libraries of compounds that can be tested for potentiating rAAV transduction. This study seeks to enhance the capability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs were tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50 to 100 times better with than without teniposide, a compound that is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy, which could result in safe and effective treatments for diverse acquired or genetic diseases.

301. AAV Capsid Engineering to Improve Transduction in Retina and Brain

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical studies for numerous disease indications including Leber's congenital amaurosis, age-related macular degeneration, hemophilia, muscular dystrophy and Parkinson's disease. AAV vectors hold considerable promise as therapeutic agents; however there is potential to further improve the efficiency of AAV gene delivery and efficacy by making modifications to the AAV capsid. The AAV capsid can be engineered to incorporate mutations that alter its transduction activity, tropism, biodistribution and immunogenicity. We have constructed variant AAV vectors harboring a variety of capsid modifications including those that negate receptor binding and have tested these vectors in several tissues including the eye and brain. One variant, AAV2HBKO, is an AAV2 based vector containing mutations of critical amino acids known to be required for binding to its receptor, heparin sulfate proteoglycan. Interestingly, an AAV2HBKO vector delivering a secreted transgene, sFLT02, unexpectedly resulted in a 2-log increase in transduction compared to parental AAV2 when delivered subretinally to the mouse eye. Subretinal delivery of an AAV2HBKO vector expressing EGFP demonstrated that these capsid modifications resulted in an increase in photoreceptor transduction compared to the unmodified AAV2 vector. In contrast, the AAV2HBKO vector demonstrated a lack of transduction activity following intravitreal delivery to the mouse eye. In addition, we evaluated the transduction and tropism of AAV2HBKO in the mouse brain. In a head to head comparison with AAV2, the AAV2HBKO vector facilitated widespread striatal and cortical expression following an intrastriatal injection while AAV2-mediated expression was restricted to the site of injection. Similar to AAV2, the tropism of AAV2HBKO was primarily neuronal with little to no transduction of astrocytes or microglia. Biodistribution data suggests that this vector, when delivered systemically in the mouse, has significantly reduced liver transduction but a higher propensity to be delivered to skeletal muscle and heart compared to the wild-type AAV2 vector. We will present data evaluating the transduction activity, tropism and biodistribution of the AAV2HBKO variant. These studies illustrate the potential for improving the efficiency of AAV gene transfer via targeted capsid engineering.