Recombinant Cells Research Articles

Optimization of the intron sequences combined with the CMV promoter increases recombinant protein expression in CHO cells

To meet the requirements of the biopharmaceutical industry, improving the yield of recombination therapeutic protein (RTP) from Chinese hamster ovary (CHO) cells is necessary. The human cytomegalovirus (CMV) promoter is widely used for RTP expression in CHO cells. To further improve RTP production, we truncated the human CMV intron and further evaluated the effect of four synthetic introns, including ctEF-1α first, EF-1α first, chimeric, and β-globin introns combined with the CMV promoter on recombinant expression levels in transient and stably recombinant CHO cells. The results showed that the truncated, EF-1α first, chimeric, and β-globin introns can significantly improve stable transgene expression in CHO cells. The qPCR results indicated that the mRNA level of transgene increased through optimizing intron sequences combined with the CMV promoter. Transcriptomics analysis was performed and found that differential expression of genes involved in mRNA processing, RNA export from nucleus, cytoplasmic translation, transcriptional activation and cell cycle regulation. In conclusion, optimization of the intron sequences combined with the CMV promoter can achieve a higher yield of recombinant proteins in CHO cells. This will be valuable for generating CHO cell lines with high productivity for industrial applications.

Comprehensive genome-scale CRISPR knockout screening of CHO cells

Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.

Enhanced Porcine Reproductive and Respiratory Syndrome Virus Replication in Nsp4- or Nsp2-Overexpressed Marc-145 Cell Lines.

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry. The killed PRRSV vaccine has been reported to be safe and could elicit humoral responses. The killed PRRSV vaccine with a high viral antigen load combined with robust adjuvants could provide good protection against the infection. However, the high virus titer of PRRSV on the successive production cell lines is the prerequisite for this strategy. In this study, we explored PRRSV production in two recombinant Marc-145 cell lines expressing Nsp2 or Nsp4 through a lentivirus system. The results demonstrated that either Nsp2 or Nsp4 expressing Marc-145 cells did not affect cell morphology and growth kinetics but significantly enhanced PRRSV replication. Overall, our exploration may enable the production of high-yield PRRSV and offer a potential tool for developing safer and more effective PRRSV vaccines.

Challenges in observing transcription–translation for bottom-up synthetic biology

Abstract Synthetic biology aims to create a viable synthetic cell. However, to achieve this goal, it is essential first to gain a profound understanding of the cellular systems used to build that cell, how to reconstitute those systems in the compartments, and how to track their function. Transcription and translation are two vital cellular systems responsible for the production of RNA and, consequently, proteins, without which the cell would not be able to maintain itself or fulfill its functions. This review discusses in detail how the Protein synthesis Using Recombinant Element (PURE) system and cell lysate are used to reconstitute transcription–translation in vitro. Furthermore, it examines how these systems can be encapsulated in GUVs using the existing methods. It also assesses approaches available to image transcription and translation with a diverse arsenal of fluorescence microscopy techniques and a broad collection of probes developed in recent decades. Finally, it highlights solutions for the challenge ahead, namely the decoupling of the two systems in PURE, and discusses the prospects of synthetic biology in the modern world.

Biocatalytic oxyfunctionalization of unsaturated fatty acids to oxygenated chemicals via hydroxy fatty acids.

The selective oxyfunctionalization of unsaturated fatty acids is difficult in chemical reactions, whereas regio- and stereoselective oxyfunctionalization is often performed in biocatalytic synthesis. Fatty acid oxygenases, including hydratases, lipoxygenases, dioxygenases, diol synthases, cytochrome P450 monooxygenases, peroxygenases, and 12-hydroxylases, are used to convert C16 and C18 unsaturated fatty acids to diverse regio- and stereoselective mono-, di-, and trihydroxy fatty acids via selective oxyfunctionalization. The formed hydroxy fatty acids or hydroperoxy fatty acids are metabolized to industrially important oxygenated chemicals such as lactones, green leaf volatiles, and bioplastic monomers, including ω-hydroxy fatty acids, α,ω-dicarboxylic acids, and fatty alcohols, by biocatalysts. For increased oxyfunctionalization of unsaturated fatty acids, enzyme engineering, functional and balanced expression in recombinant cells, selection of suitable catalyst types, and reaction engineering have been suggested. This review describes biocatalysts involved in the oxyfunctionalization of unsaturated fatty acids and the production of hydroxy fatty acids and oxygenated chemicals.

Conjugate Position of Glucuronide and Sulfate in Piceatannol Derivatives Affects the Stability and Hydrolytic Resistance of the Conjugate in Biological Matrices.

Piceatannol, a stilbene compound, undergoes a comprehensive phase II metabolism mediated by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in humans. Despite their well-documented beneficial effects on health, their detailed pharmacokinetic fate, including the metabolite structure and properties, is poorly understood. Thus, we determined the structure of seven glucuronides and six sulfates transformed from piceatannol and its methylated derivatives in recombinant yeast cells expressing UGTs or SULTs. We evaluated their properties in human and rat plasma samples. The conjugate that was substituted at the 3'- or 4'-catecholic hydroxy moiety exhibited increased stability. The sulfatase-mediated hydrolysis assay results in incomplete digestion or compound degradation of certain sulfates, suggesting a potential risk of underestimation by using indirect quantification methods. These findings emphasize the importance of an authentic standard for accurate pharmacokinetic studies of phase II metabolites that will be useful for understanding the mechanisms underlying the functional contribution of piceatannol in the body.

Cell line development and bioreactor process optimization for an atezolizumab biosimilar.

Checkpoint inhibitors are widely recognized immunotherapeutic drugs, known for their effectiveness in treating various cancers. Atezolizumab, targeting the immune checkpoint programmed death-ligand 1, is successfully used to treat several types of cancers. Atezolizumab is a potential biosimilar candidate due to its huge success in the clinic but there is no literature on its production process in mammalian cells. In this study, we generated a monoclonal cell line derived from recombinant Chinese hamster ovary DG44 cells to produce atezolizumab. The selected single clone was employed for media screening and process development. Following production in a 7-L bioreactor, atezolizumab was purified using a three-step chromatographic method. Finally, the purified atezolizumab was characterized and compared with commercial atezolizumab (Tecentriq) through several chromatographic and kinetics analyses.

Optimum blue light exposure: a means to increase cell-specific productivity in Chinese hamster ovary cells

Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering.Key points• Blue LED light hinders growth in CHO DP-12 cells• Antioxidants protect to a certain degree from blue light effects• Illumination with blue LED light raises cell-specific productivity

Chemically Synthesized 1,2,3,4,6-Pentakis-O-Galloyl-β-D-Glucopyranoside Blocks SARS-CoV-2 Spike Interaction with Host ACE-2 Receptor.

In the search for anti-COVID-19 therapy, 1,2,3,4,6-pentakis-O-galloyl-βD-glucopyranoside, a natural polyphenolic compound isolated from many traditional medicinal herbs, has been reported as an RBD-ACE2 binding inhibitor and as a broad-spectrum anticoronaviral inhibitor targeting the main protease and RNA-dependent RNA polymerase of SARSCoV-2. To facilitate the structure-activity relationship studies of 1,2,3,4,6-pentakis-O-galloyl-β-Dglucopyranoside, we describe its chemical synthesis and characterization, as well as its activity towards the SARS-CoV-2 spike interaction with host ACE2 receptor. 1,2,3,4,6-Pentakis-O-galloyl-β-D-glucopyranoside was synthesized in two quantitative steps from 3,4,5-tribenzyloxybenzoic acid and β-D-glucopyranoside: DCC-mediated esterification and palladium-catalyzed per-debenzylation. The synthesized molecule was evaluated using a SARS-CoV-2 spike trimer (S1 + S2) ACE2 inhibitor screening colorimetric assay kit, SARS-CoV2 spike S1 RBD ACE2 inhibitor screening colorimetric assay kit, and a cellular neutralization assay using the Spike (SARS-CoV-2) Pseudotyped Lentivirus, ACE2-HEK293 recombinant cell line. The chemically synthesized product blocked the binding of the spike trimer of SARSCoV-2 to the human ACE2 receptor with IC50=22±2 µM. It also blocked ACE2:spike RBD binding with IC50=27±3 µM. Importantly, it inhibited the infectivity of SARS2-CoV2-Spike pseudotyped lentivirus on the ACE2 HEK293 cell line with IC50=20±2 µM. Overall, the chemically synthesized 1,2,3,4,6-pentakis-O-galloyl-β-D-glucopyranoside represents a lead molecule to develop anti-SARS-CoV-2 therapies that block the initial stage of the viral infection by blocking the virus entry to the host cell.

Persistent Rhesus Enteric Calicivirus Infection in Recombinant CHO Cells Expressing the Coxsackie and Adenovirus Receptor.

Recently, using a panel of recombinant CHO cell lines, we identified the coxsackie and adenovirus receptor (CAR) and histo-blood group antigens (HBGAs) or sialic acid as the minimum requirement for susceptibility to rhesus enteric calicivirus (ReCV) infections. While ReCVs cause lytic infection in LLC-MK2 cells, recombinant CHO (rCHO) cell lines did not exhibit any morphological changes upon infection. To monitor infectious virus production, rCHO cell cultures had to be freeze-thawed and titrated on LLC-MK2 monolayers. This raised the question of whether ReCV infection in rCHO cells is persistent and whether non-enveloped progeny virions are released from the infected cells. Here, we used the rCHO-CAR+ cell line and a CAR and sialic acid-dependent recovirus strain (FT7) and found that these cells were persistently infected, and infectious virus was continuously produced and released into the culture without showing any visible cell damage. Viral capsid protein and replication intermediate double-stranded RNA (dsRNA) were detectable in almost all cells for at least 12 passages. We suspect a fully exosomal viral exit mechanism without a lytic cycle in these cells. rCHO cell may provide a valuable system for ReCV production (producer cell line) and serve as a model for investigating enteric calicivirus non-lytic viral exit mechanisms and the properties of the released, most likely membrane-cloaked, infectious progeny virions.

Single dose of recombinant baculovirus vaccine expressing sigma B and sigma C genes provides good protection against novel duck reovirus challenge in ducks

The novel duck reovirus (NDRV) disease causes high economic losses, resulting in substantial economic losses in waterfowl industry. However, currently, no commercial vaccines are available to alleviate NDRV infection throughout the world. Here, we developed two subunit vaccine candidates for NDRV based on the insect cell-baculovirus expression system (IC-BEVS). Two recombinant viruses, namely rBac-σB and rBac-σC, were successfully generated based on the consensus sequence of NDRV. Then, the σB and σC subunit vaccine candidates were prepared by directly inactivating the recombinant virus infected-Sf9 cell suspension. The double antibody-sandwich ELISA was used for quantitative of σB or σC protein in the inactivated crude antigen. Protective efficacy results revealed that, compared with the whole virus inactivated vaccine, a single dose of 160 ng σB or σC protein showed advantages in inducing serum antibodies, elevating weight, alleviating liver and spleen injury, restraining viral shedding and viral replication in ducklings. To be noted, the subunit σC or the combination of subunit σB and σC vaccine candidates had better protective efficacies, especially the combined σB and σC vaccine group. Therefore, our study provides useful information for developing effective vaccine against NDRV infection.

Expression of Viral DNA Polymerase in Synthetic Recombinant Adeno-Associated Virus Producer Cell Line Enhances Full Particle Productivity.

Recombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 106 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV-1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV-1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

A Lipase Gene of Thermomyces lanuginosus: Sequence Analysis and High-Efficiency Expression in Pichia pastoris.

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application.

Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. There have been relatively few studies on the phosphoproteome of recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for phosphoproteomic analysis of CHO cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using liquid chromatography mass spectrometry (LC-MS), multistage activation (MSA), and MS3 strategies. This protocol can also be used for quantitative phosphoproteomic analysis using both labeled and label-free approaches.

Recent Advancements in Proteomic Sample Preparation from Recombinant Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and post-translationally modify recombinant proteins that are compatible with human use. Proteomics, along with other "omic platforms," are being used to understand the biology of CHO cells with the ultimate aim to enhance CHO cell factories for more efficient production of biopharmaceuticals. Liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis has become the standard technique for profiling proteomes. There are three stages to a typical bottom-up approach, namely, sample preparation, LC-MS/MS, and data analysis. Although there have been major advances in LC-MS/MS instrumentation and data analysis tools, sample preparation is still the crucial stage that affects the overall efficiency of any proteomic study. In this chapter, we present a comparison of a number of widely used sample preparation methods for proteomic applications, including in-solution digestion and commercially available device-based kits. All methods performed well; however, each method has inherent advantages and disadvantages.

Splicing by Overlap Extension PCR for the Production of Fusion Proteins.

Fusion proteins are valuable molecules to meet different demands related to the development of biopharmaceuticals and bioprocesses. In human therapy, they are used to improve the half-life of biologics by modifying the biophysical properties of the proteins. In biotechnology, the design of fusion proteins can standardize the establishment of production clones and the purification process. Analytical detection capabilities of the fusion partner and binding to affinity ligands play a crucial role. For the generation of the recombinant cell line, the maturation of the protein and the secretion are also crucial factors, which can be significantly influenced by the fusion partner and candetermine the final yield of the bioprocess. Here we present a protocol to recombine the human extracellular domain of CD19 with the human serum albumin domain 2 resulting in a fusion protein CD19-AD2 including a flexible linker sequence in the interface between the C-terminus of CD19 and the N-terminus of HSA-D2 and a terminal His12-tag. The two fragments are independently amplified with primers allowing to genetically connect the two fragments in the next step by overlap extension PCR. By this strategy, the linker sequence as well as the albumin fragment can be chosen individually to be flexible in the fine-tuning of the final protein. The amplified product is then cloned into a mammalian expression vector suitable to generate a recombinant transient or stable cell culture. This workflow can be applied to any protein sequence by adapting the specific primer sequences.

Downregulation of CD86 in HCMV-infected THP-1 cells.

Monocytes and macrophages are at the frontline of defense against pathogens. Human cytomegalovirus (HCMV) uses myeloid cells as vehicles to facilitate viral dissemination. HCMV infection in monocytes and macrophages leads to the downregulation of several cell surface markers via an undefined mechanism. Previously, we showed that HCMV pUL42 associates with the Nedd4 family ubiquitin E3 ligases through the PPXY motif on pUL42 and downregulates Nedd4 and Itch proteins in HCMV-infected fibroblasts. Homologous proteins of HCMV pUL42, such as HHV-6 U24, downregulate cell surface markers. To reveal the downregulation property of pUL42, we focused on CD86, the key costimulatory factor for acquired immunity. Here, we constructed CD86-expressing THP-1 cells using a retroviral vector and analyzed the effects of HCMV infection and pUL42 on CD86 downregulation. Disruption of the PPXY motifs of pUL42 (UL42PA) decelerated the degradation of CD86 in recombinant virus-infected cells, indicating the involvement of Nedd4 family functions. However, no direct interactions were observed between CD86 and Itch. Interestingly, unlike fibroblast infection, the expression of Nedd4 and Itch proteins increased in HCMV-infected THP-1 cells, accompanied by an increase in their transcript levels. Although the function of pUL42 did not relate to the increase of Nedd4 and Itch proteins, pUL42 should affect these Nedd4 proteins to downregulate CD86.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

Introducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

Utilizing Adenovirus Knob Proteins as Carriers in Cancer Gene Therapy Amidst the Presence of Anti-Knob Antibodies.

Numerous gene therapy drugs for cancer have received global approval, yet their efficacy against solid tumors remains inadequate. Our previous research indicated that the fiber protein, a component of the adenovirus capsid, can propagate from infected cells to neighboring cells that express the adenovirus receptor. We hypothesize that merging this fiber protein with an anti-cancer protein could enable the anti-cancer protein to disseminate around the transfected cells, presenting a novel approach to cancer gene therapy. In our study, we discovered that the knob region of the adenovirus type 5 fiber protein is the smallest unit capable of spreading to adjacent cells in a receptor-specific manner. We also showed that the recombinant knob protein infiltrates cells after dispersing to surrounding cells. To assess the potential of the knob protein to augment gene therapy for solid tumors in mice, we expressed a fusion gene of the A subunit of cytotoxic cholera toxin and the knob region in mouse tumors. We found that this fusion protein only inhibited tumor growth in receptor-expressing mouse melanomas, and this inhibitory effect persisted even in mice with anti-knob antibodies. Our study's findings propose a novel cancer gene therapy strategy that enhances therapeutic effects by specifically delivering therapeutic proteins, expressed from in vivo administered genes, to target molecules. This outcome offers a fresh perspective on gene therapy for solid cancers, and we anticipate that knob proteins will serve as a platform for this method.