Adeno-associated Virus Infection Research Articles

Role of the membrane-associated accessory protein (MAAP) in adeno-associated virus (AAV) infection.

Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation

ObjectiveProinflammatory necroptosis is the main pathological mechanism of ischemic stroke. Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffolding protein that exerts anti-inflammatory effects in most central nervous system diseases. However, the relationship between Homer1 and proinflammatory necroptosis in ischemic stroke remains unclear.AimThis study aimed to investigate the role of Homer1 in ischemia-induced necroptosis.MethodsC57BL/6 mice were used to establish a model of permanent middle cerebral artery occlusion model (pMCAO). Homer1 knockdown mice were generated using adeno-associated virus (AAV) infection to explore the role of Homer1 and its impact on necroptosis in pMCAO. Finally, Homer1 protein was stereotaxically injected into the ischemic cortex of Homer1flox/flox/Nestin-Cre +/− mice, and the efficacy of Homer1 was investigated using behavioral assays and molecular biological assays to explore potential mechanisms.ResultsHomer1 expression peaked at 8 h in the ischemic penumbral cortex after pMCAO and colocalized with neurons. Homer1 knockdown promoted neuronal death by enhancing necroptotic signaling pathways and aggravating ischemic brain damage in mice. Furthermore, the knockdown of Homer1 enhanced the expression of proinflammatory cytokines. Moreover, injection of Homer1 protein reduced necroptosis-induced brain injury inhibited the expression of proinflammatory factors, and ameliorated the outcomes in the Homer1flox/flox/Nestin-Cre+/− mice after pMCAO.ConclusionsHomer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation. These data suggested that Homer1 is a novel regulator of neuronal death and neuroinflammation.

Circular RNA circARPC1B functions as a stabilisation enhancer of Vimentin to prevent high cholesterol-induced articular cartilage degeneration.

Osteoarthritis (OA) is a prevalent and debilitating condition, that is, directly associated with cholesterol metabolism. Nevertheless, the molecular mechanisms of OA remain largely unknown, and the role of cholesterol in this process has not been thoroughly investigated. This study aimed to investigate the role of a novel circular RNA, circARPC1B in the relationship between cholesterol and OA progression. We measured total cholesterol (TC) levels in the synovial fluid of patients with or without OA to determine the diagnostic role of cholesterol in OA. The effects of cholesterol were explored in human and mouse chondrocytes in vitro. An in vivo OA model was also established in mice fed a high-cholesterol diet (HCD) to explore the role of cholesterol in OA. RNAseq analysis was used to study the influence of cholesterol on circRNAs in chondrocytes. The role of circARPC1B in the OA development was verified through circARPC1B overexpression and knockdown. Additionally, RNA pulldown assays and RNA binding protein immunoprecipitation were used to determine the interaction between circARPC1B and Vimentin. CircARPC1B adeno-associated virus (AAV) was used to determine the role of circARPC1B in cholesterol-induced OA. TC levels in synovial fluid of OA patients were found to be elevated and exhibited high sensitivity and specificity as predictors of OA diagnosis. Moreover, elevated cholesterol accelerated OA progression. CircARPC1B was downregulated in chondrocytes treated with cholesterol and played a crucial role in preserving the extracellular matrix (ECM). Mechanistically, circARPC1B is competitively bound to the E3 ligase synoviolin 1 (SYVN1) binding site on Vimentin, inhibiting the proteasomal degradation of Vimentin. Furthermore, circARPC1B AAV infection alleviates Vimentin degradation and OA progression caused by high cholesterol. These findings indicate that the cholesterol-circARPC1B-Vimentin axis plays a crucial role in OA progression, and circARPC1B gene therapy has the opportunity to provide a potential therapeutic approach for OA.

Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.

The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.

AAV induces hepatic necroptosis and carcinoma in diabetic and obese mice dependent on Pebp1 pathway.

Obesity and diabetes are risk factors for hepatocellular carcinoma (HCC); however, the underlying mechanisms are yet to be elucidated. Adeno-associated virus (AAV) frequently infects humans and has been widely used in gene therapy, but the risk of AAV infection such as HCC should be further evaluated. Here, we show that recombinant AAV injection caused liver injury, hepatic necroptosis, and HCC in db/db or high-fat diet-induced hyperglycemic and obese mice, but not in mice with only hyperglycemia or obesity. Prednisone administration or knockdown of Pebp1, highly expressed in db/db mice, alleviated hepatic injury and necroptosis induced by recombinant AAV in mice with diabetes and obesity. Inhibition of Pebp1 pathway also attenuated inflammation and necroptosis in vitro. Our findings show that AAV infection is a critical risk factor for HCC in patients with diabetes and obesity, and AAV gene therapy for these patients should be carefully evaluated. Both prednisone treatment and targeting Pebp1 pathway are promising strategies to alleviate inflammation and necroptosis that occurred in AAV gene therapy or related diseases.

In situ-formable, dynamic crosslinked poly(ethylene glycol) carrier for localized adeno-associated virus infection and reduced off-target effects

The adeno-associated virus (AAV) is a potent vector for in vivo gene transduction and local therapeutic applications of AAVs, such as for skin ulcers, are expected. Localization of gene expression is important for the safety and efficiency of genetic therapies. We hypothesized that gene expression could be localized by designing biomaterials using poly(ethylene glycol) (PEG) as a carrier. Here we show one of the designed PEG carriers effectively localized gene expression on the ulcer surface and reduced off-target effects in the deep skin layer and the liver, as a representative organ to assess distant off-target effects, using a mouse skin ulcer model. The dissolution dynamics resulted in localization of the AAV gene transduction. The designed PEG carrier may be useful for in vivo gene therapies using AAVs, especially for localized expression.

A Single Surface-Exposed Amino Acid Determines Differential Neutralization of AAV1 and AAV6 by Human Alpha-Defensins.

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors due to their low pathogenicity and tissue tropism properties. However, the efficacy of these vectors is impeded by interactions with the host immune system. One potential immune barrier to vector transduction is innate immune host defense peptides, such as alpha-defensins, which are potent antiviral agents against other nonenveloped viruses. To investigate the interaction between AAVs and alpha-defensins, we utilized two closely related AAV serotypes, AAV1 and AAV6. Although their capsids differ by only six residues, these two serotypes exhibit markedly different tissue tropisms and transduction efficiencies. Using two abundant human alpha-defensins, enteric human defensin 5 (HD5) and myeloid human neutrophil peptide 1 (HNP1), we found both serotype-specific and defensin-specific effects on AAV infection. AAV6 infection was uniformly neutralized by both defensins at low micromolar concentrations; however, inhibition of AAV1 infection was profoundly influenced by the timing of defensin exposure to the virus relative to viral attachment to the cell. Remarkably, these differences in the defensin-dependent infection phenotype between the viruses are completely dictated by the identity of a single, surface-exposed amino acid (position 531) that varies between the two serotypes. These findings reveal a determinant for defensin activity against a virus with unprecedented precision. Furthermore, they provide a rationale for the investigation of other AAV serotypes not only to understand the mechanism of neutralization of defensins against AAVs but also to design more efficient vectors. IMPORTANCE The ability of adeno-associated viruses (AAVs) to infect and deliver genetic material to a range of cell types makes them favorable gene therapy vectors. However, AAV vectors encounter a wide variety of host immune factors throughout the body, which can impede efficient gene delivery. One such group of factors is the alpha-defensins, which are a key component of the innate immune system that can directly block viral infection. By studying the impact that alpha-defensins have on AAV infection, we found that two similar AAV serotypes (AAV1 and AAV6) have different sensitivities to inhibition. We also identified a single amino acid (position 531) that differs between the two AAV serotypes and is responsible for mediating their defensin sensitivity. By investigating the effects that host immune factors have on AAV infection, more efficient vectors may be developed to evade intervention by the immune system prior to gene delivery.

Consideration on the possible etiological mechanisms and countermeasures about severe acute hepatitis of unknown origin in children

Since April 2022, severe acute hepatitis of unknown origin in children has spread to 35 countries and regions around the world, and more than 1 010 cases have been reported. Since the severe acute hepatitis of unknown origin involves a wide range of areas and has a high rate, it is critical to identify the etiology and establish effective preventive, diagnostic and therapeutic measures as soon as possible. This study discusses the possible mechanisms and countermeasures of the severe acute hepatitis of unknown origin in children. It speculates that the occurrence of the recent severe acute hepatitis might be related to adenovirus, adeno-associated virus infection, and the COVID-19 epidemic, while the difference in HLA polymorphism among different races might be related to the fact that reported cases were more common in Europe and the United States. Based on the currently available evidence, it can be preliminarily judged that the risk of large-scale outbreak of severe acute hepatitis of unknown origin in children would be low in China, but the persistent awareness and vigilance of the etiology is still needed.

AAV infection of bovine embryos: Novel, simple and effective tool for genome editing

Adeno-associated viruses (AAV) are widely used in the field of genetically modified organism production. In this work, transduction of bovine embryos by AAV was selected as a potential approach to perform genetic modifications: we have used recombinant AAV to produce GFP-positive bovine embryos. Five different AAV serotypes were used to evaluate their ability to deliver genetic material into the bovine embryos. AAV9 serotype demonstrated minimal effectiveness (38,10%) as the genetic material transfer tool. Four other serotypes of AAVs (AAV1, AAV2, AAV6 and AAV-DJ) showed very close transduction efficiency (52,94–58,33%). CD209 is a C-type lectin receptor which is presented on the surface of macrophages and dendritic cells. CD209 recognizes a broad range of pathogens in a rather nonspecific manner. Production of CD209 knock-out is relevant for better understanding of infection mechanisms. Potentially, production of such knock-out may enable animals to become resistant to various infections. We have analyzed DNA samples from 22 blastocysts obtained after in vitro culture of zygotes subjected to recombinant AAV action. We have detected that 3 of 22 analyzed blastocysts contained mosaic CD209 frameshifts. Therefore, we have demonstrated proof of principle that application of AAV as a genome editing tool is an effective method for obtaining genetically modified cattle embryos.

Macrophage Distribution Affected by Virus-Encoded Granulocyte Macrophage Colony Stimulating Factor Combined with Lactate Oxidase.

Oncolytic virotherapy was approved as a localized treatment for advanced melanoma by the US Food and Drug Administration (FDA) in 2015. Granulocyte macrophage colony stimulating factor (GM-CSF) encoded by clinical virus-infected tumor cells, acting as a pro-inflammatory cytokine or growth factor, increases tumor antigen presentation, leading to the activation of macrophages and T cells. Notably, tumor-secreted lactate can promote the suppressive functions of M2-polarized tumor-associated macrophages and subsequently promote tumor growth. Furthermore, the consumption of tumor-secreted lactate has been implicated in the beneficial polarization of macrophages. Here, we report that GM-CSF-encoded recombinant adeno-associated virus (AAV2-GM-CSF) infection in B16-F10 mouse melanoma cells combined with lactate oxidase (LOX) leads to the recruitment of M1 macrophages for the inhibition of cancer cell growth. This study suggests that GM-CSF combined with LOX has potential as cancer virotherapy.

Natural antisense RNA Foxk1-AS promotes myogenic differentiation by inhibiting Foxk1 activity

BackgroundNatural antisense RNAs are RNA molecules that are transcribed from the opposite strand of either protein-coding or non-protein coding genes and have the ability to regulate the expression of their sense gene or several related genes. However, the roles of natural antisense RNAs in the maintenance and myogenesis of muscle stem cells remain largely unexamined.MethodsWe analysed myoblast differentiation and regeneration by overexpression and knockdown of Foxk1-AS using lentivirus and adeno-associated virus infection in C2C12 cells and damaged muscle tissues. Muscle injury was induced by BaCl2 and the regeneration and repair of damaged muscle tissues was assessed by haematoxylin–eosin staining and quantitative real-time PCR. The expression of myogenic differentiation-related genes was verified via quantitative real-time PCR, Western blotting and immunofluorescence staining.ResultsWe identified a novel natural antisense RNA, Foxk1-AS, which is transcribed from the opposite strand of Foxk1 DNA and completely incorporated in the 3′ UTR of Foxk1. Foxk1-AS targets Foxk1 and functions as a regulator of myogenesis. Overexpression of Foxk1-AS strongly inhibited the expression of Foxk1 in C2C12 cells and in tibialis anterior muscle tissue and promoted myoblast differentiation and the regeneration of muscle fibres damaged by BaCl2. Furthermore, overexpression of Foxk1-AS promoted the expression of Mef2c, which is an important transcription factor in the control of muscle gene expression and is negatively regulated by Foxk1.ConclusionThe results indicated that Foxk1-AS represses Foxk1, thereby rescuing Mef2c activity and promoting myogenic differentiation of C2C12 cells and regeneration of damaged muscle fibres.DfdN9uwC-a513-TBDoKHuCVideo

Dichotomous Roles of Men1 in Macrophages and Fibroblasts in Bleomycin-Induced Pulmonary Fibrosis.

Pulmonary fibrosis therapy is limited by the unclear mechanism of its pathogenesis. C57BL/6 mice were used to construct the pulmonary fibrosis model in this study. The results showed that Men1, which encodes menin protein, was significantly downregulated in bleomycin (BLM)—induced pulmonary fibrosis. Mice were made to overexpress or had Men1 knockdown with adeno-associated virus (AAV) infection and then induced with pulmonary fibrosis. BLM—induced pulmonary fibrosis was attenuated by Men1 overexpression and exacerbated by Men1 knockdown. Further analysis revealed the distinct roles of Men1 in fibroblasts and macrophages. Men1 inhibited fibroblast activation and extracellular matrix (ECM) protein expression while promoting macrophages to be profibrotic (M2) phenotype and enhancing their migration. Accordingly, pyroptosis was potentiated by Men1 in mouse peritoneal macrophages (PMCs) and lung tissues upon BLM stimulation. Furthermore, the expression of profibrotic factor OPN was positively regulated by menin in Raw264.7 cells and lung tissues by binding to the OPN promoter region. Taken together, although Men1 showed antifibrotic properties in BLM—induced pulmonary fibrosis mice, conflictive roles of Men1 were displayed in fibroblasts and macrophages. The profibrotic role of Men1 in macrophages may occur via the regulation of macrophage pyroptosis and OPN expression. This study extends the current pathogenic understanding of pulmonary fibrosis.

MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies

Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG1–3 with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.

Mesenchymal stem cells inhibited the apoptosis of alveolar epithelial cells caused by ARDS through CXCL12/CXCR4 axis

ABSTRACT Mesenchymal stem cells (MSCs) have a wide range of anti-inflammatory and immunomodulatory effects and have been observed to have potential therapeutic potential in the clinical treatment of various diseases. We pretreated lung cancer cells A549 with tumor necrosis factor (TNF-α), knocked down the key chemokine receptor CXCR4 on MSCs using lentivirus, and induced acute respiratory distress syndrome (ARDS) mouse model using lipopolysaccharide (LPS) and CXCL12 expression in vivo by adeno-associated virus (AAV-rh10) infection in mice. By co-culturing the MSCs with A549 and in vivo experiments, we observed the effects of MSCs on cell biological functions after inflammatory stimulation, oxidative stress, and the amelioration of lung injury in ARDS mice. TNF-α inhibited A549 proliferation and promoted apoptosis, scorch death-related factor activity, and oxidative stress factor were increased and CXCL12 levels in the cell supernatant were decreased. The co-culture of MSCs was able to increase cell activity and decrease oxidative stress factor levels, and this effect was not present after the knockdown of CXCR4 in MSCs. In vivo transplantation of MSCs significantly attenuated lung injury in ARDS, inhibited serum pro-inflammatory factors in mice, and down-regulated expression of apoptotic and focal factors in lung tissues while blocking CXCR4 or CXCL12 lost the repairing effect of MSCs on ARDS lung tissues. After the co-culture of MSC and lung cancer cells, the expression of CXCR4 on the surface of lung cancer cells was significantly increased, and more CXCR4 and CXCL12 acted together to activate more pro-survival pathways and inhibit apoptosis induced by TNF-α.

Homer1 promotes the conversion of A1 astrocytes to A2 astrocytes and improves the recovery of transgenic mice after intracerebral hemorrhage

BackgroundInflammation induced by intracerebral hemorrhage (ICH) is one of the main causes of the high mortality and poor prognosis of patients with ICH. A1 astrocytes are closely associated with neuroinflammation and neurotoxicity, whereas A2 astrocytes are neuroprotective. Homer scaffolding protein 1 (Homer1) plays a protective role in ischemic encephalopathy and neurodegenerative diseases. However, the role of Homer1 in ICH-induced inflammation and the effect of Homer1 on the phenotypic conversion of astrocytes remain unknown.MethodsFemoral artery autologous blood from C57BL/6 mice was used to create an ICH model. We use the A1 phenotype marker C3 and A2 phenotype marker S100A10 to detect astrocyte conversion after ICH. Homer1 overexpression/knock-down mice were constructed by adeno-associated virus (AAV) infection to explore the role of Homer1 and its mechanism of action after ICH. Finally, Homer1 protein and selumetinib were injected into in situ hemorrhage sites in the brains of Homer1flox/flox/Nestin-Cre+/− mice to study the efficacy of Homer1 in the treatment of ICH by using a mouse cytokine array to explore the potential mechanism.ResultsThe expression of Homer1 peaked on the third day after ICH and colocalized with astrocytes. Homer1 promotes A1 phenotypic conversion in astrocytes in vivo and in vitro. Overexpression of Homer1 inhibits the activation of MAPK signaling, whereas Homer1 knock-down increases the expression of pathway-related proteins. The Homer1 protein and selumetinib, a non-ATP competitive MEK1/2 inhibitor, improved the outcome in ICH in Homer1flox/flox/Nestin-Cre+/− mice. The efficacy of Homer1 in the treatment of ICH is associated with reduced expression of the inflammatory factor TNFSF10 and increased expression of the anti-inflammatory factors activin A, persephin, and TWEAK.ConclusionsHomer1 plays an important role in inhibiting inflammation after ICH by suppressing the A1 phenotype conversion in astrocytes. In situ injection of Homer1 protein may be a novel and effective method for the treatment of inflammation after ICH.

Response Selectivity of the Lateral Posterior Nucleus Axons Projecting to the Mouse Primary Visual Cortex

Neurons in the mouse primary visual cortex (V1) exhibit characteristic response selectivity to visual stimuli, such as orientation, direction and spatial frequency selectivity. Since V1 receives thalamic visual inputs from the lateral geniculate nucleus (LGN) and lateral posterior nucleus (LPN), the response selectivity of the V1 neurons could be influenced mostly by these inputs. However, it remains unclear how these two thalamic inputs contribute to the response selectivity of the V1 neurons. In this study, we examined the orientation, direction and spatial frequency selectivity of the LPN axons projecting to V1 and compared their response selectivity with our previous results of the LGN axons in mice. For this purpose, the genetically encoded calcium indicator, GCaMP6s, was locally expressed in the LPN using the adeno-associated virus (AAV) infection method. Visual stimulations were presented, and axonal imaging was conducted in V1 by two-photon calcium imaging in vivo. We found that LPN axons primarily terminate in layers 1 and 5 and, to a lesser extent, in layers 2/3 and 4 of V1, while LGN axons mainly terminate in layer 4 and, to a lesser extent, in layers 1 and 2/3 of V1. LPN axons send highly orientation- and direction-selective inputs to all the examined layers in V1, whereas LGN axons send highly orientation- and direction-selective inputs to layers 1 and 2/3 but low orientation and direction selective inputs to layer 4 in V1. The distribution of preferred orientation and direction was strongly biased toward specific orientations and directions in LPN axons, while weakly biased to cardinal orientations and directions in LGN axons. In spatial frequency tuning, both the LPN and LGN axons send selective inputs to V1. The distribution of preferred spatial frequency was more diverse in the LPN axons than in the LGN axons. In conclusion, LPN inputs to V1 are functionally different from LGN inputs and may have different roles in the orientation, direction and spatial frequency tuning of the V1 neurons.

High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy.

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.

Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein\u2013Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV\u2009+\u2009lymphoproliferative disorders

BackgroundB-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein–Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible.MethodsAs a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay.ResultsRank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir.ConclusionsrAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis

BackgroundEngineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3’ end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results.ResultsWe collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients.By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported.We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients.ConclusionsOur findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

Functional roles of the membrane-associated AAV protein MAAP

With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.