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Schematic representation of the vectors containing different 2A peptides for mAb expression (A) and corresponding amino acid sequences of various 2A peptides (B). The conserved regions of 2A peptides are highlighted in dotted boxes. CMV, human cytomegalovirus IE gene promoter; HC, heavy chain cDNA; Furin, DNA encoding furin recognition sequence RRKR; LC, light chain cDNA; IRESatt, mutated encephalomyocarditis virus (EMCV) internal ribosomal entry site; DHFR, dihydrofolate reductase cDNA; SpA, simian virus 40 early polyadenylation signal.
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Abstract Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2...
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Citations
... cDNAs encoding mAbs of interest were synthesized (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) or Fab expression vector (designated as pTwist-mCis_FAB) and used for production in mammalian cell culture. This vector contains an enhanced 2A sequence and GSG linker that allows for the simultaneous expression of mAb heavy-and light-chain genes from a single construct upon transfection 79 . For antibody production, we performed transfection of ExpiCHO cell cultures using the Gibco ExpiCHO Expression System as described by the vendor. ...
Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.
... Our new transgenic line, coupled with a rapid and simple detection method to detect tau spread, remedies these limitations. Our transgene, expressed under the control of the Q system (30), consists of the coding sequences of human tau and GFP with an intervening T2A protein cleavage sequence (31). GFP, which is cleaved from tau during translation and does not spread, marks the expression sites of our constructs; the spread of tau can thus be detected as locations where tau is present but GFP is not. ...
... Second, to visualize tau spread while simultaneously marking the original site of expression, we generated a QF2-responsive construct consisting of the coding sequence of human tau tagged with FLAG, followed by a T2A protein self-cleavage sequence (31) and then by GFP (Fig. 1A). ...
Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau in Alzheimer’s disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We find age-dependent tau spread into the brain, represented by detection of tau, but not GFP in the brain. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin or the kinase glycogen synthase kinase-3β ( GSK-3β ). Further work revealed that dynamin promotes tau uptake in recipient tissues, whereas GSK-3β appears to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau trafficking components involved in the pathogenesis of neurodegenerative diseases.
SUMMARY STATEMENT
The trafficking of toxic proteins in neurodegenerative disease is well-known but poorly understood. Our model will allow rapid and new insight into molecular mechanisms underlying this process.
... For example, the use of 2A self-cleaving peptides allows the generation of two proteins from a single mRNA transcript due to ribosomal skipping during translation. 8 However, this requires the presence of a 2A peptide sequence on the coding sequence, which may not always be possible or desirable. Moreover, this system always leads to an equimolar ratio of the subunits. ...
... Alternatively, on the mRNA level, internal ribosome entry sites (IRES) allow translation initiation from more than one open-reading frame (ORF) on the same primary transcript, 9 but at a fixed and non-tuneable ratio of subunits. In general, polycistronic approaches lead to lower titers (below 1 g/ L) 8,10 compared to monocistronic approaches with titers in the multigram per liter range. ...
The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.
... These two NA proteins were separated by a P2A sequence. Theoretically, when the lipid nanoparticle (LNP) carrying the Influenza A N1 + N2 mRNA enters host cells and undergoes successful translation, the ribosome would undergo "self-cleavage" between the glycine and proline residues at the C-terminus of the P2A sequence [28], resulting in the separation of the two NA proteins and their independent folding into individual proteins. However, due to the lack of suitable antibodies for detecting these two NA proteins, there is no direct evidence to suggest that these two NA proteins can form homologous or heterologous tetramers in vivo, as designed. ...
Vaccines are one of the most effective means of preventing influenza A, typically containing the hemagglutinin (HA) of the influenza A virus. However, antigenic drift and shift of the influenza A virus can lead to instability in vaccine efficacy. Compared to HA, the antigenic variation rate of neuraminidase (NA) is slower. In traditional inactivated influenza vaccines, although they contain a certain amount of NA, there are significant differences between different batches, which cannot consistently induce NA-based immune responses. Therefore, NA is often overlooked in vaccine development. In this study, we report an mRNA vaccine encoding the NA of two strains of influenza A virus. The experimental results demonstrated that when matched with the viral strain, this mRNA vaccine induced high levels of neutralizing antibodies, providing a protective effect to mice in viral challenge experiments, and this immune response was shown to be biased towards the Th1 type. In summary, this study demonstrates that NA is a promising potential antigen, providing new insights for the development of influenza A virus vaccines.
... We chose NanoLuc luciferase as our second reporter protein, which includes an N-terminal IL-6 secretion signal, offering a highly sensitive and cost-efficient readout from culture supernatant at multiple time points ( Figure 1D) [37]. We connected both reporter proteins via a linker region containing a Furin cleavage site, a V5-Tag, and a P2A site for simultaneous and equimolar protein expression and analysis as previously described [38]. ...
... We chose NanoLuc luciferase as our second reporter protein, which includes an N-terminal IL-6 secretion signal, offering a highly sensitive and cost-efficient readout from culture supernatant at multiple time points ( Figure 1D) [37]. We connected both reporter proteins via a linker region containing a Furin cleavage site, a V5-Tag, and a P2A site for simultaneous and equimolar protein expression and analysis as previously described [38]. can also be monitored via the NanoLuc expression level through luminescence analysis. ...
Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies.
... The amino acid sequence of the furin-2A site [61] was as follows: RRKRGSGE-GRGSLLTCGDVEENPGP. ...
... The VZV antigen we selected was the full-length gE protein, the target of protective immunity in the highly efficacious Shingrix vaccine. Antigens were encoded in a single-expression cassette and linked via a furin-2A sequence [61] designed to result in translation of two separate, conformationally correct antigens. Two ChAdOx2 vectors were generated: one encoding RSV preF upstream of VZV gE (ChAdOx2-RSV-VZV) and vice versa (ChAdOx2-VZV-RSV), to enable selection of the best-performing vector. ...
Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.
... This is achieved by the transduction of long-lived cells with a viral vector engineered to express the heavyand light-chain domains of an mAb [4]. These domains are separated by a foot-and-mouth disease virus 2A self-cleaving peptide sequence containing a furin cleavage site at the amino terminus to ensure optimal processing [5]. The singular polyprotein produced in vivo should be indistinguishable from those produced by the endogenous immune system [6]. ...
... A recent follow-up study carried out by Gardner et al. aimed to further optimize the AAV VIP delivery system [33]. Many studies examining the expression of HIV, Ebola virus, and Marburg virus antibodies have all included an F2A peptide sequence; however, previous work has indicated that porcine teschovirus-1 2A (P2A) and Thosea asigna virus 2A (T2A) peptides might be more efficient alternatives [5,53]. The researchers first evaluated transgene cassette design, comparing the efficiencies of P2A, F2A, and T2A self-cleaving peptides expressed within the sequence of the anti-SIV antibody ITS01 by an AAV9 vector in mice. ...
Monoclonal antibodies (mAbs) are important treatment modalities for preventing and treating infectious diseases, especially for those lacking prophylactic vaccines or effective therapies. Recent advances in mAb gene cloning from naturally infected or immunized individuals has led to the development of highly potent human mAbs against a wide range of human and animal pathogens. While effective, the serum half-lives of mAbs are quite variable, with single administrations usually resulting in short-term protection, requiring repeated doses to maintain therapeutic concentrations for extended periods of time. Moreover, due to their limited time in circulation, mAb therapies are rarely given prophylactically; instead, they are generally administered therapeutically after the onset of symptoms, thus preventing mortality, but not morbidity. Adeno-associated virus (AAV) vectors have an established record of high-efficiency in vivo gene transfer in a variety of animal models and humans. When delivered to post-mitotic tissues such as skeletal muscle, brain, and heart, or to organs in which cells turn over slowly, such as the liver and lungs, AAV vector genomes assume the form of episomal concatemers that direct transgene expression, often for the lifetime of the cell. Based on these attributes, many research groups have explored AAV-vectored delivery of highly potent mAb genes as a strategy to enable long-term expression of therapeutic mAbs directly in vivo following intramuscular or intranasal administration. However, clinical trials in humans and studies in nonhuman primates (NHPs) indicate that while AAVs are a powerful and promising platform for vectored immunoprophylaxis (VIP), further optimization is needed to decrease anti-drug antibody (ADA) and anti-capsid antibody responses, ultimately leading to increased serum transgene expression levels and improved therapeutic efficacy. The following review will summarize the current landscape of AAV VIP in NHP models, with an emphasis on vector and transgene design as well as general delivery system optimization. In addition, major obstacles to AAV VIP, along with implications for clinical translation, will be discussed.
... To develop an RGC-specific inducible Cre mouse line, we chose to insert a P2A-CreER T2 gene cassette in in-frame fusion to the C-terminus of RBPMS, an RNA splicing protein known to be expressed in all RGCs ( Figure 1A). The addition of the highly efficient P2A self-cleaving peptide allows the co-expression of RBPMS and CreER T2 from a single transcript due to ribosomal skipping during protein translation [60] without disrupting the function of RBPMS. CreER T2 , a Cre recombinase fused to a mutant human estrogen ligand-binding domain, permits the nuclear translation and activation of Cre recombinase activity upon binding of synthetic ligands, such as 4-hydroxytamoxifen and tamoxifen [61]. ...
Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain, and dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing for the co-expression of RBPMS and CreERT2. The inducible Rbpms-CreERT2 mice exhibited a high recombination efficiency in activating the expression of the tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, both heterozygous and homozygous Rbpms-CreERT2 knock-in mice showed no detectable defect in the retinal structure, visual function, and transcriptome. Together, these results demonstrated that the Rbpms-CreERT2 knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study, and ocular disease modeling in RGCs.
... Other plant produced proteins expressed showed yields of 18.49 mg/kg of human interleukin-6 58 , 3 mg/kg of cocaine-hydrolase Fc protein 59 , 730 mg/kg of CMG2-Fc protein 60 . These short time periods provide essential advantages of protein expression in terms of speed over other expression approaches including mammalian cells [61][62][63][64] and stable plant expression [65][66][67] systems. The significant reduction in antibody expression after 5 dpi might be due to the progressive development of necrosis in the infiltrated leaves. ...
As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC’s by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.
... We constructed a mouse anti-Scg3 mAb Fab fragment in an AAV2 vector with a CAG promoter [28]. The heavy and light chains contained the same human/murine IgG heavy chain signal peptide, separated by a furin GT2A cleavage site ( Figure 1A) [24,25]. A FLAG tag was attached to the C-terminus of the light chain. ...
... Safety concerns related to chronic VEGF suppression prompted the testing of multiple strategies to regulate anti-VEGF gene therapy [63][64][65]. Because our studies indicate that Scg3 regulates only pathological angiogenesis, with no effect on healthy vessels or neurons [24,26], such safety issues may not apply, and long-term constitutive gene therapy with anti-Scg3 hAb is expected to be safe. Our findings warrant further investigation to compare the efficacy and safety of optimized AAV-anti-Scg3 and AAV-anti-VEGF for monotherapy or combination therapy in large animal models. ...
Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.
