AAV Infection Research Articles

Metalloparticle-Engineered Pickering Emulsion Displaying AAV-Vectored Vaccine for Enhancing Antigen Expression and Immunogenicity Against Pathogens.

Recombinant adeno-associated viruses (rAAVs) have emerged as promising vaccine vectors due to their enduring efficacy with a single dose. However, insufficient cellular immune responses and the random and non-specific distribution of AAVs post-injection may hinder the development of AAV vaccines. Here, a novel Pickering emulsion platform stabilized by biomineralized manganese nanoparticles and aluminum hydroxide, which can rapidly and efficiently load AAVs, is reported. This platform confers AAVs with favorable in vivo distribution kinetics, diversifying AAV endocytic pathways with reduced dependency on the sialic acid receptor-mediated route, and ultimately enhancing AAV infection efficiency in antigen present cells (APCs). Concurrently, the Pickering emulsion substantially boosts endogenous 2'3'-cGAMP production, further activating the cGAS-STING pathway for stronger immune responses and improving protective efficacy in bacterial infection models. The STING pathway activation also increases AAV target gene expression, potently augmenting the cross-protective potential of AAV vaccines for COVID-19. These synergistic effects ensure that effective immune responses are induced even at one-fifth of the AAV vaccination dose, while the Pickering emulsion further reduces the accumulation of AAV in the liver, thereby improving their safety. The findings highlight the potential of Pickering emulsions as valuable enhancers for viral vectors, providing insights for their broader clinical applicability.

Mechanisms of AAV2 neutralization by human alpha-defensins.

AAVs are commonly used as gene therapy vectors due to their broad tropism and lack of disease association; however, host innate immune factors, such as human alpha-defensin antimicrobial peptides, can hinder gene delivery. Although it is becoming increasingly evident that human alpha-defensins can block infection by a wide range of nonenveloped viruses, including AAVs, their mechanism of action remains poorly understood. In this study, we describe for the first time how two types of abundant human alpha-defensins neutralize a specific AAV serotype, AAV2. We found that one defensin prevents AAV2 from binding to cells, the first step in infection, while both defensins block a critical later step in AAV2 entry. Our findings support the emerging idea that defensins use a common strategy to block infection by DNA viruses that replicate in the nucleus. Through understanding how innate immune effectors interact with and impede AAV infection, vectors can be developed to bypass these interventions and allow more efficient gene delivery.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

Development of Assays to Measure GNE Gene Potency and Gene Replacement in Skeletal Muscle.

GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gne gene expression with human GNE in skeletal muscles. A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNE gene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. Lec3 cells showed a strong deficit in MAA binding, while GNE-/-MB135 cells did not. Overexpressing GNE in Lec3 and Lec3MyoDI cells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNE stimulated human GNE expression in muscles at levels equivalent to endogenous mouse Gne at a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. Lec3 and Lec3MyoDI cells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNE can deliver GNE gene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.

CircZSWIM6 mediates dysregulation of ECM and energy homeostasis in ageing chondrocytes through RPS14 post-translational modification.

Circular RNAs (CircRNAs) are important and have different roles in disease progression. Herein, we aim to elucidate the roles of a novel CircRNA (CircZSWIM6) which is upregulated in ageing chondrocytes. We verified the roles of CircZSWIM6 in senescent and osteoarthritis (OA) development in vitro through CircZSWIM6 knockdown and overexpression. RNA pulldown assay and RNA binding protein immunoprecipitation were performed to identify the interaction between CircZSWIM6 and Ribosomal protein S14 (RPS14). The roles of CircZSWIM6 in ageing-related OA were also confirmed in non-traumatic and traumatic model respectively. CircZSWIM6 regulates extracellular matrix (ECM) and energy metabolism in ageing chondrocyte. Mechanistically, CircZSWIM6 competitively bound to the E3 ligase STUB1 binding site on RPS14 (K125) to inhibit proteasomal degradation of RPS14 to maintain RPS14 function. CircZSWIM6-RPS14 axis is highly associated with AMPK signaling transduction, which keeps energy metabolism in chondrocyte. Furthermore, CircZSWIM6 AAV infection leads to senescent and OA phenotypes in a non-traumatic model and accelerates OA progression in a traumatic model. Our results revealed a significant role of CircZSWIM6 in age-related OA by regulating ECM metabolism and AMPK-associated energy metabolism. We highlight the CircZSWIM6-RPS14-PCK1-AMPK axis is a potential biomarker for OA.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Adeno-associated virus in the liver: natural history and consequences in tumour development

ObjectiveAdeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%–80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed...

PGC-1/ERR-Induced Regulator in Muscle (PERM1) Increases Mitochondrial Respiratory Capacity in Culture Muscle Cells

Peripheral arterial disease is the third leading cause of death of atherosclerotic cardiovascular mortality in the United states and the incidence of PAD increases age. Previous studies have shown that PAD displays impaired mitochondrial respiration, decreased expression of mitochondrial enzymes, increased oxidative stress, and mitochondrial DNA mutations within their ischemic limb muscles. We identified a potential transcriptional regulator of mitochondrial gene expression, PGC-1/ERR-induced regulator in muscle (PERM1) which is reduced 85% in patients with severe PAD. Interestingly, PERM1 regulates the expression of only a subset of genes induced by PGC-1a or ERRs expression in C2C12 myotubes, suggesting that PERM1 selectively functions in specific PGC-1/ERR-driven pathways. PURPOSE: The purpose of this study is to determine whether PERM1 is a potential gene target to aid in tissue recovery and regeneration from hypoxia in C2C12 myotubes. METHODS: We generated AAVs to overexpress the PERM1 gene or a green fluorescent protein (ZsGreen1). Following AAV infection, C2C12 myoblasts were differentiated into mature myotubes for assessments of mitochondrial biogenesis, mitochondrial respiration, and myogenesis were performed. To determine if PERM1 plays a role in recovering myotube respiration and myotube atrophy from hypoxia, C2C12 myotubes infected AAV-PERM1 were placed in hypoxia for 6-hours and mitochondrial respiratory function, content, and myosin heavy chain area were assessed during the recovery from hypoxic insult. RESULTS: AAV-PERM1 resulted in a ~16-fold increase in mRNA expression which drove a ~20% increase in complex I-supported respiration compared to the control cells (P<0.05, n=6). Increased Expression of PERM1 did not change the myotube fusion index (an indicator of myogenesis) (P=0.32, n=4). CONCLUSION: Our results indicate that PERM1 is a strong regulator of mitochondrial biogenesis in skeletal muscle cells, capable of increase both mitochondrial content and respiratory function. Based on these observations, future studies should be aimed at understanding the therapeutic potential of PERM1 for improving muscle energetics in conditions with impaired muscle metabolism, including peripheral arterial disease. Partially supported by AHA Grant 18CDA34110044 to TER.

Viral Delivery of GFP-Dependent Recombinases to the Mouse Brain.

Many genetic tools have been developed that use green fluorescent protein (GFP) and its derivatives for labeling specific cell populations in organisms and in cell culture. To extend the use of GFP beyond labeling purposes, we developed methods and reagents that use GFP as a driver of biological activities. We used nanobodies that bind GFP to engineer CRE-DOG and Flp-DOG, recombinases that can induce Cre/lox and Flp/FRT recombination in a GFP-dependent manner, respectively. Here, we present a protocol to deliver CRE-DOG and Flp-DOG into the mouse brain by recombinant AAV infection. This protocol enables one to manipulate gene expression specifically in GFP-expressing cells, found either in transgenic GFP reporter lines or in cells made to express GFP by other transduction methods.

AAV Infection: Protection from Cancer.

There are conflicting reports that integration of the wild-type adeno-associated virus 2 (AAV2) genome is associated with induction of hepatocellular carcinoma (HCC) in a small subset of patients. However, there are several lines of evidence that contradict this assertion: (i) AAV2 has long been known to be a non-pathogenic virus, although ∼90% of the human population is seropositive for AAV2 antibodies; (ii) AAV2 has been shown to possess anticancer activity; (iii) epidemiological evidence suggests that AAV2 infection plays a protective role against cervical carcinoma; and (iv) five different AAV serotype vectors (AAV1, AAV2, AAV5, AAV8, and AAV9) have been or are currently being used in 162 Phase I/II clinical trials and one Phase III clinical trial in humans to date, and no cancer of any type has ever been observed or reported. A brief historical account of the putative role of infection by AAV in the etiology of cancer, or lack thereof, is presented.

550. Developing an Optimal Insulin Augmentation Therapy to Improve AAV Gene Transfer to Skeletal Muscle and Liver in Mice

We previously reported a novel finding that showed an insulin augmentation therapy can improve rAAV transduction of skeletal muscle and liver in cultured cells and mice. The insulin augmentation therapy in this earlier report was given just prior to AAV administration and was sustained for 28 days. The use of a long-term insulin augmentation was to determine if mice showed early signs of insulin resistance due to prolonged hyperinsulinemia. From the initial study we concluded that long-term insulin augmentation therapy improved AAV gene transfer to skeletal muscle and liver but that the mice showed a lack of elevated insulin activity and an increase in cortisol over time which are signs of insulin resistance. Additionally, our initial findings also suggested that an acute insulin augmentation therapy during the window of AAV administration would be sufficient without the concern for developing insulin resistance. The purpose of this new study was to investigate if an acute insulin augmentation therapy during the window of AAV delivery was sufficient to improve AAV transduction of skeletal muscle and liver. Additionally, we wanted to determine if an insulin augmentation therapy given after AAV infection would also enhance AAV gene transfer. Another aspect of this follow up study was to determine if an insulin augmentation therapy protocol would improve AAV transduction of lung tissue in mice. Finally, we wanted to investigate if fasting mice (which promotes reduced insulin activity) would result in sub-optimal AAV gene transfer. Our results showed an acute insulin augmentation therapy (4 hr treatment) given 30 minutes prior to intramuscular (IM) injection of AAV1-CMV-sc-hFIX or liver-directed delivery (via splenic capsule injection) of AAV8-CMV-sc-hFIX significantly improved (up to 5 Fold) sustained transduction for the length of the study (56 days).View Large Image | Download PowerPoint SlideThe acute therapy showed a minor (but significant) 1.7 Fold increase in AAV1-CMV-EGFP transduction in lung. In-vitro AAV2-CMV-LacZ transduction of HuH7 (human liver) and differentiated C2C12 (mice myofibers) also showed improvement with an acute insulin treatment. A 2hr treatment (5 ug/ml) resulted in a 2.7 fold increase while 8hr, 24hr & 72hr treatments had a 3.5 fold increase and giving insulin 24 hrs after AAV infection had no impact of AAV transduction.View Large Image | Download PowerPoint SlideFinally, prolonged fasting will reduce insulin levels which could impact AAV gene transfer. Fasting mice for 12 hrs prior to and 4 hrs after IM delivery of AAV1-CMV-sc-hFIX or at weekly intervals after administration timed around blood collection did not have an impact on AAV transduction of skeletal muscle.

467. Assay Conditions Significantly Alter AAV Neutralizing Antibody Determination

A major limitation of vectors based on current AAVs is the wide prevalence of memory B and T cells reactive to the AAV capsid in human populations arisen following natural AAV infection. The Neutralizing Antibody (NAB) assay guides experimentation and determines inclusion or exclusion of subjects in clinical studies in order to predict in vivo neutralization. Furthermore, evidence of a memory B-cell response to AAV may correlate with anti-capsid memory T-cell populations implicated in the safety and efficacy outcome of clinical AAV liver studies. Based on previously reported findings, NAB assay protocols in current use are unable to robustly predict in vivo neutralization in monkeys (Zinn et al. Cell Reports, 2015 & Wang et al. Mol Ther, 2010) leading to false negatives. In a clinical setting, the enrollment of a seropositive individual exposes the subject to undue risk, and generates outcome variability of the often small clinical studies. Here, we sought to permutate several of the NAB assay conditions in order to evaluate their impact on the NAB titer readout. Specifically, we varied the following variables: cell line, transgene detection, use of adenovirus, and AAV amount. Prior to evaluating these assay permutation, we sought to increase the sensitivity of the readout for transgene expression in the assay in order to evaluate at all conditions both high and low transducing AAV serotypes. This optimization increased our sensitivity by ~200-fold. With this increased sensitivity of readout, we first sought to evaluate the compliance of standard AAVNAB assays with the percentage law, formulated by Andrewes and Elford in 1933, a standard for virological NAB assays, which seeks stoichiometric conditions of NAB and viral load that enables measurement of NAB titers independent of viral input in the assay. Our data shows that at the conditions in common use in the field the percentage law is not upheld, and NAB titers are highly dependent on input viral load of the assay, directly impacting sensitivity and specificity, and leading to the occurrence of false negatives. Indeed, quantitative assessment of seroprevalence in primate populations with assays that reduce the amount of AAV to within percentage law range affects assay outcome qualitatively and quantitatively. We also independently assessed other assay parameters to improve sensitivity, robustness and reproducibility, and to study whether they may also have qualitative effects on titer outcomes. Our studies highlight the importance of pre-existing immunity in AAV gene therapy and limitations of the current methodologies to measure it in a robust and predictive manner. Data indicates the potential for false positive and negative readout for commonly used assay protocols. An optimized protocol was developed for further validation and evaluation. These studies may impact translational and clinical AAV gene therapy studies.

Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU).

The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca2 + uptake. Cytosolic Ca2 + transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2 + regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2 + transients elicit large increases in the [Ca2 +] of the mitochondrial matrix ([Ca2 +]mt). Mitochondrial Ca2 + uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2 + uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2 + uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931).

38. Pharmacological Regulation of Vesicular Trafficking as a Strategy to Enhance Recombinant AAV Transduction

Vector dose-related toxicity has been highlighted as a potential safety concern in recent preclinical studies and clinical trials with recombinant AAV vectors. Thus, investigation of strategies to enhance AAV transduction may allow for administration of lower vector doses, thereby mitigating toxicity. Several host cell factors have been shown to restrict viral infection during post-entry trafficking steps through a variety of mechanisms. In the case of AAV, the 26S proteasome is thought to reduce transduction efficiency by restricting nuclear entry via ubiquitin-dependent capsid degradation. The AAA+ ATPase p97/valosin-containing protein (VCP) is a segregase and unfoldase which is closely associated with the proteasomal degradation pathway and disassembles multimeric protein complexes for subsequent processing. VCP modulates numerous cellular processes ranging from endosomal sorting to autophagy and ER-associated degradation. In the current study, we sought to determine whether VCP plays a role as a restriction factor in the AAV infectious pathway. Chemical inhibition of VCP by Eeyarestatin I (EerI) increased AAV transduction by nearly an order of magnitude in a cell type and serotype independent manner. This effect was associated with increased recovery of viral genomes and capsid accumulation within the nucleus, but not cell surface binding or uptake. Interestingly, VCP appears to restrict AAV infection by regulating vesicular trafficking of viral particles. In particular, VCP inhibition redistributes AAV capsids to Rab7 and LAMP1 positive vesicles, while simultaneously disrupting capsid association with syntaxin-5 positive vesicles recently shown to be essential for Golgi transport. These results support the notion that AAV vesicular transport can be redirected to enhance transduction efficiency. In summary, our results identify VCP as a novel host restriction factor that regulates the post-entry trafficking of recombinant AAV vectors, and highlight VCP as a potential target for modulating AAV transduction in the clinic.

61. Whole Genome, High Throughput Functional Screening for Cellular Factors Regulating AAV Infection Identifies the ERI-1 Exoribonuclease as a Powerful Inducer of AAV Vector Transduction

61. Whole Genome, High Throughput Functional Screening for Cellular Factors Regulating AAV Infection Identifies the ERI-1 Exoribonuclease as a Powerful Inducer of AAV Vector Transduction

Mapping the AAV Capsid Host Antibody Response toward the Development of Second Generation Gene Delivery Vectors.

The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

Mitotic Catastrophe Occurs in the Absence of Apoptosis in p53-Null Cells with a Defective G1 Checkpoint

Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions in which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death of p53-defective cells in mitosis, without affecting the integrity of the host genome. Osteosarcoma cells (U2OSp53DD) that are deficient in p53 and lack the G1 cell cycle checkpoint respond to AAV infection through a transient G2 arrest. We found that the infected U2OSp53DD cells died through mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. These findings were confirmed by time-lapse microscopy of cellular morphology following AAV infection. The assays used readily revealed apoptosis in other cell types when it was indeed occurring. Taken together the results indicate that in the absence of the G1 checkpoint, mitotic catastrophe occurs in these p53-null cells predominantly as a result of mechanical disruption induced by centrosome overduplication, and not as a consequence of a suicide signal.

Adenovirus–Adeno-Associated Virus Hybrid for Large-Scale Recombinant Adeno-Associated Virus Production

Recombinant adeno-associated virus (rAAV) holds promise for applications in gene therapy. Advances in clinical studies of rAAV-based gene therapeutics have generated an encouraging momentum in the field of gene therapy; however, one of the major obstacles to the eventual clinical success of rAAV-mediated gene therapy is the need for large-scale production of clinical-grade vectors. The transfection-based rAAV production method is well suited for preclinical studies in small animal models, but it is difficult to support large-scale clinical studies with this method. In the past decade, several scalable rAAV production methods have emerged from extensive efforts to develop large-scale manufacturing processes. Among those, the recombinant adenovirus-AAV infection method has some unique features in vector quality and yield. This minireview provides an overview of this scaleable rAAV production platform, describing its basic components and biological mechanisms and process.

Transduction efficiency of AAV 2/6, 2/8 and 2/9 vectors for delivering genes in human corneal fibroblasts

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1 × 10 5 and 2 × 10 5 MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 h after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6 ⋙ AAV2/9 > AAV2/8. The transduction efficiency of AAV2/6 was 30–50-fold higher ( p < 0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4 h was considerably low compared to 30 h suggesting that the transgene delivery did not reach its peak at 4 h. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.

115. Whole Genome Analysis Shows That AAV Integrates at Chromosome 19 AAV-S1 Sites and Other Chromosomal Sites That Frequently Share GGTC Micro-Homology with the AAV P5 Promoter

Adeno associated virus undergoes Rep-mediated integration into the AAV-S1 region of human chromosome 19 during latent infection but recombinant AAV (rAAV) vectors are missing essential viral components and lack such site-specificity. This was best shown recently by strategies that monitor rAAV integration events at the level of the whole eukaryotic genome. However, whole-genome analyses have not been used to analyse integration by wild-type AAV. We have used ligation-mediated PCR (LM-PCR) to monitor AAV integration under both latent and replication-permissive conditions of infection. Sequence analysis of fifty-two clones that met pre-determined criteria showed that, during replication, AAV integrated into many chromosomal sites via sequence microhomology that frequently involved a GGTC motif located within the AAV p5 element, with one site preferred. No clones containing specific AAV/AAV-S1 junctions were identified among authentic LM-PCR clones, although such clones were readily obtained using junction-specific PCR primers; suggesting that AAV-S1 integration events constituted only a small portion of the total integration product. During latent AAV infection, GGTC-mediated integration was significantly reduced, but such micro-homology-determined chromosomal junctions were still obtained in excess over AAV-S1 specific products. This new mechanism for the integration of AAV sequences into human chromosomes has a |[ldquo]|signature|[rdquo]| that suggests it may occur independently of Rep, although the detailed mechanism remains to be elucidated. Finally, since others have shown that the p5 element is necessary and sufficient for AAV-S1 chromosomal integration, in view of our new findings, the specificity of new rAAV vectors that include p5-region sequences for improved Repmediated integration should be closely monitored, especially by whole-genome approaches.