Figure 10 - available from: Scientific Reports
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MAAP trans-complementation. We performed wt-AAV2 and MAAP-S33-S39-S47 variant production with or without the addition of recombinant MAAP. Vg titers (vg mL −1 ) were measured from cell extract samples harvested 24 hpt (A) and 72 hpt (B). Graphics show individual samples with mean and SD. Statistical significance was evaluated for recombinant MAAP addition using ANOVA followed by Dunnett's multiple comparison test. Tables (C) and (D) represent the average titer (vg mL −1 ) at 24 hpt and 72 hpt, with fold difference to wt-AAV2. The samples from left to right are: (1) wt-AAV2. (2) MAAP-S33-S39-S47. (3) wt-AAV2 trans-complemented with MAAP expressing plasmid. (4) MAAP-S33-S39-S47 trans-complemented with MAAP expressing plasmid. (5) wt-AAV2 trans-complemented with a GFP plasmid of similar size to the recombinant MAAP plasmid. (6) MAAP-S33-S39-S47 complemented with a GFP plasmid of similar size to the recombinant MAAP plasmid.
Source publication
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...
Context in source publication
Context 1
... wanted to know how recombinant MAAP overproduction affects wt-AAV2 and MAAP-S33-S39-S47 variant titers. Compared to plain wt-AAV2 production, the overproduction caused viral titers to decrease by 33% at 24 and 64% at 72 hpt ( Fig. 10A-D). A GFP control plasmid of similar size as MAAP plasmid reduced the viral titers only by 9% at 24 hpt and 4% at 72 hpt. When MAAP-S33-S39-S47 was applied to virus production, the titers increased 1.65-fold at 24 hpt and 5.89-fold at 72 hpt. MAAP overexpression in combination with MAAP-S33-S39-S47, however, resulted in similar vg titers ...
Citations
... VP1, VP2, and VP3 are encoded by the cap gene, which form the capsid that encapsidates the viral genome to form the virus particle (Maurer and Weitzman 2020;Muhuri et al. 2021). Assembly-activating protein (AAP) and membrane-associated accessory protein (MAAP), involved in AAV capsid assembly and secretion respectively, are also encoded by the cap gene (Sonntag et al. 2011;Elmore et al. 2021;Galibert et al. 2021). ...
... MAAP has been reported to be expressed from the + 1 frameshifted open reading frame in the VP1 region of the AAV cap gene, which may be in the spliced form of the p40 transcript that encodes VP2/3 and AAP (Elmore et al. 2021;Galibert et al. 2021). Thus, MAAP transcript was not Fig. 1 Schematic diagram of three AAV production systems. ...
Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines.
Key points
• wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2.
• Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2.
• wtAAV infection and rAAV production in GX2 elicited similar host cell responses.
... AAV8 MAAP (MAAP8) has been observed in the promotion of secreting produced vectors by interacting with extracellular vesicles (37). In cells infected with adenovirus (Ad), transfection of a full-length AAV2 genome clone demonstrated that MAAP2 functions as an accelerator for AAV2 replication and likely in viral genome packaging (34). However, the exact roles of the MAAP during AAV infection and its expression strategy remain elusive, and nothing is known about the expression of AAV5 MAAP (MAAP5) and its function. ...
... MAAP ablation increases the total production of progeny viruses, which is likely related to the increase in viral DNA replication. The basic-amino-acid-rich (BR) presence of the MAAP at the C-terminus may function as a nuclear localization (NLS) (34) and lead to nuclear localization of the MAAP. It is interesting that the nuclear expression of MAAP5 was much more obvious than that of MAAP2, which perhaps is related to the extended 17 aa at the N-terminus. ...
... Without MAAP expression, fewer progeny virions are egressed out of the infected cells, which may result in more virions stuck in the nucleus and thus more viral DNA templates for replication. However, others have observed that MAAP ablation or early termination decreased AAV2 production in an AAV2 infectious (34). We believe that this discrepant observation is probably attributable to the lytic infection system employed by them. ...
Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection.
IMPORTANCE
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.
... These are integral in capsid assembly as well as distribution of the virus throughout cellular compartments. (Galibert et al., 2021;Sonntag et al., 2010). The wild-type REP and CAP genes can be replaced with a transgenic construct, but the ITRs must remain for the ssDNA to be successfully loaded into a capsid. ...
... Together, these three genes mediate genome replication and packaging, capsid production, and integration [10,11]. More recently, an additional gene product coding for a membrane-associated accessory protein (MAAP) has been identified that facilitates AAV egress and encapsulation [12,13]. ...
Gene therapy holds promise for patients with inherited monogenic disorders, cancer, and rare genetic diseases. Naturally occurring adeno-associated virus (AAV) offers a well-suited vehicle for clinical gene transfer due to its lack of significant clinical pathogenicity and amenability to be engineered to deliver therapeutic transgenes in a variety of cell types for long-term sustained expression. AAV has been bioengineered to produce recombinant AAV (rAAV) vectors for many gene therapies that are approved or in late-stage development. However, ongoing challenges hamper wider use of rAAV vector-mediated therapies. These include immunity against rAAV vectors, limited transgene packaging capacity, sub-optimal tissue transduction, potential risks of insertional mutagenesis and vector shedding. This review focuses on aspects of immunity against rAAV, mediated by anti-AAV neutralizing antibodies (NAbs) arising after natural exposure to AAVs or after rAAV vector administration. We provide an in-depth analysis of factors determining AAV seroprevalence and examine clinical approaches to managing anti-AAV NAbs pre- and post-vector administration. Methodologies used to quantify anti-AAV NAb levels and strategies to overcome pre-existing AAV immunity are also discussed. The broad adoption of rAAV vector-mediated gene therapies will require wider clinical appreciation of their current limitations and further research to mitigate their impact.
... The cap gene encodes the three VP1, VP2, and VP3 proteins, which constitute the viral capsid and the membrane-associated accessory protein (MAAP) which aids AAV replication and has a role in controlling HAdV infection. The assembly of the capsid is facilitated by the assembly activating protein (AAP) [148,[181][182][183]. Many host cell surface molecules are recognised by infecting AAV-2 (reviewed [184]). ...
Over 100 human adenoviruses (HAdVs) have been isolated and allocated to seven species, A-G. Species F comprises two members-HAdV-F40 and HAdV-F41. As their primary site of infection is the gastrointestinal tract they have been termed, with species A, enteric adenoviruses. HAdV-F40 and HAdV-F41 are a common cause of gastroenteritis and diarrhoea in children. Partly because of difficulties in propagating the viruses in the laboratory, due to their restrictions on growth in many cell lines, our knowledge of the properties of individual viral proteins is limited. However, the structure of HAdV-F41 has recently been determined by cryo-electron microscopy. The overall structure is similar to those of HAdV-C5 and HAdV-D26 although with some differences. The sequence and arrangement of the hexon hypervariable region 1 (HVR1) and the arrangement of the C-terminal region of protein IX differ. Variations in the penton base and hexon HVR1 may play a role in facilitating infection of intestinal cells by HAdV-F41. A unique feature of HAdV-F40 and F41, among human adenoviruses, is the presence and expression of two fibre genes, giving long and short fibre proteins. This may also contribute to the tropism of these viruses. HAdV-F41 has been linked to a recent outbreak of severe acute hepatitis “of unknown origin” in young children. Further investigation has shown a very high prevalence of adeno-associated virus-2 in the liver and/or plasma of some cohorts of patients. These observations have proved controversial as HAdV-F41 had not been reported to infect the liver and AAV-2 has generally been considered harmless.
... In addition, a region in the cap vp1 gene encodes the non-structural membrane-associated AAV protein (MAAP), which is a potential AAV egress factor [9][10][11]. A second region overlapping all the cap genes encodes the assembly activating protein (AAP). Transcription of the AAP mRNA is initiated by a non-canonical start codon (CTG) [12]. ...
... Monoclonal antibodies are advantageous, because of their defined properties and reproducibility, yet easy to obtain polyclonal sera are still used. MAAP of AAV2 Anti-MAAP GAL-KKI polyclonal 79-98 of MAAP [11] In AAV research, the mouse monoclonal antibody (mAb) A20 has been established in early work in the group of Kleinschmidt along with anti-VP mAbs A69, B1 and anti-Rep mAbs 76/3 (also named 76.3) and 303/9 [80]. A20 is the prototype antibody only recognizing assembled capsids of AAV serotype 2. The same group demonstrated the use of these antibodies in fluorescence microscopy to track subcellular location during transduction and AAV formation in combination with DNA in situ hybridization [43]. ...
... Galibert et al. detected MAAP with a custom generated polyclonal antibody generated toward a MAAP peptide (MAAP-GALKKI antibody) followed by an anti-rabbit AlexaFluorconjugated secondary antibody [11]. ...
Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.
... The absence of at least the last 10 amino acids at the C-terminus of the MAAP protein reduced the degradation of the capsid and increased the expression of capsid proteins, while other truncated forms of MAAP completely prevented the degradation of the AAV2 capsid, which led to a 3.5-fold increase in assembled capsids for some variants. It can be assumed that MAAP somehow affects the processes of capsid degradation and the stability of VPs [94]. It also can be assumed that, since truncated forms or inactivated MAAP leads to increased expression of the AAP, this protein directly leads to the prevention of degradation of VPs and an increase in their stability. ...
... Only the stable truncated form of MAAP at the C-terminus showed levels of contamination similar to that of the wild type. In addition, this variant contributed to an increased level of viral particles containing the AAV genome [94]. Thus, MAAP plays a significant role in the biology of AAV and the production of viral particles, thereby representing a promising object for research and optimization of the assembly process. ...
The adeno-associated virus (AAV) is one of the most potent vectors in gene therapy. The experimental profile of this vector shows its efficiency and accepted safety, which explains its increased usage by scientists for the research and treatment of a wide range of diseases. These studies require using functional, pure, and high titers of vector particles. In fact, the current knowledge of AAV structure and genome helps improve the scalable production of AAV vectors. In this review, we summarize the latest studies on the optimization of scalable AAV production through modifying the AAV genome or biological processes inside the cell.
... The rep gene is required for viral genome replication and packaging, the cap gene produces viral capsids, the aap gene promotes capsid assembly, and the maap gene helps facilitate viral replication. 24,25 Conversely, rAAV does not contain these genes and only requires the presence of 130 bp AAV ITR arms flanking a DNA fragment of up to 4.9 kb on either side for packaging. 26 The ITRs are the only cis-acting components necessary for the packaging and replication of DNA fragments. ...
... Vectors were titered by droplet digital PCR (ddPCR) and ELISA as described previously (34). In ddPCR, viral genomes were quantified with CMV promoter primers (Supplementary Table 3). ...
Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B . Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3′ end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.
... Three structural capsid proteins (VP1, VP2 and VP3), encoded by the cap gene, are also expressed and these form the viral capsid with help from the assembly activating protein (AAP). A fourth ORF encodes the membrane-associated accessory protein (MAAP) [15,[60][61][62]. VP3, the major subunit of the capsid, is responsible for tissue tropism through cell receptor recognition [63,64]. ...
Over the past few months there have been reports of severe acute hepatitis in several hundred, otherwise healthy, immunocompetent young children. Several deaths have been recorded and a relatively large proportion of the patients have needed liver transplants. Most of the cases, so far, have been seen in the UK and in North America, but it has also been reported in many other European countries, the Middle East and Asia. Most common viruses have been ruled out as a causative agent; hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were not detected, nor were Epstein–Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in many cases. A small proportion of the children had been infected with SARS-CoV-2 but these seem to be in a minority; similarly, almost none of the children had been vaccinated against COVID-19. Significantly, many of the patients were infected with adenovirus 41 (HAdV-F41). Previously, HAdV-41 had not been linked to hepatitis and is usually considered to cause gastroenteritis in both immunocompetent and immunocompromised patients. In two most recent studies, adeno-associated virus 2 (AAV2) was detected in almost all patients, together with species C and F HAdVs and human herpesvirus 6B (HHV6B). Here, I discuss the possibility that a change in tropism of HAdV-41 and changes in AAV2 may be responsible for their links to acute hepatitis.
