Although cutaneous melanoma accounts for only about 2% of skin cancers, its rapid progression makes it an aggressive skin cancer with a high mortality rate. As of 2018, the SEER database estimated that the 5-year overall survival (OS) rate is 29.8% in patients with stage IV disease at diagnosis in the United States. Non-cutaneous melanoma, including mucosal and uveal subtypes, carries a generally worse prognosis. Once considered refractory to conventional treatments, such as chemotherapy and radiation therapy, the advent of immunotherapy, including immune checkpoint inhibitors (ICIs), vaccines, and tumor-infiltrating lymphocytes (TIL), and of targeted therapy over the past decade has resulted in dramatic improvements in melanoma. Importantly, ICIs have resulted in long-term remission for patients with melanoma, thus introducing the possibility of a cure for some patients with metastatic disease. These include antibodies against programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), and lymphocyte activation gene-3 (LAG-3). In this review, we will provide an overview of metastatic melanoma while focusing on its current pharmacologic armamentarium, toxicities of treatment, including ICIs and targeted therapy, and its therapeutic clinical strategies. The therapeutic advances presented in this review serve as the foundation for an ever-expanding repertoire of innovative approaches. These include mRNA vaccines, oncolytic viruses, bispecific engagers, oral immunomodulators, and novel cytokines. Adoptive cellular strategies are evolving to TILS transduced with conditional gene expression cassettes, as well as non-T cell approaches involving dendritic cells and natural killer (NK) cells. Targeted therapy strategies have broadened to include upstream components of RAS, other MAP kinase pathways, and HDAC inhibitors, among others. All these new paradigms translate into increasingly complex decision-making for the treatment team, a burden that is more than offset by the tremendous benefit for melanoma patients. This is truly the beginning of a new era.

Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.

Adeno-associated virus (AAV), a 4.7kb single-stranded DNA virus, is widely used as a gene therapy vector, but its limited packaging capacity poses challenges for delivering large genes, always resulting in truncation during packaging. Quantifying truncation is difficult because both strands of the plasmid can be packaged from the 3' end. In this article, we aim to first produce single-polarity AAV and then explore its truncation pattern. To address this, we modified one of the ITRs and created an oversized self-complementary AAV, which functions as a single-polarity vector to some extent. Using this modified backbone, we generated a series of oversized single-polarity AAV (spAAV) vectors of varying lengths and sequences, analyzing DNA truncation patterns via quantitative PCR (qPCR). The results show that as the distance from the 3'-ITR increased, less DNA was detected. At 3000bp from the 3'-ITR, 70% of the genomic DNA remained; this dropped to 50% at 4000bp, 20% at 4500bp, and almost none beyond 5000bp. Additionally, reporter gene expression significantly decreased when the expression cassette extended to 4.5kb compared to 2.7kb under identical in vitro conditions. Our results show that DNA will be truncated far earlier before 4.5kb during the packaging of very large genomes. This study provides important insights into the truncation patterns of AAV genomes, which is crucial for optimizing AAV vector design in gene therapy.

The nonconventional yeast, Yarrowia lipolytica, is a promising protein expression host, having achieved recombinant protein expression yield on par with the commonly used host, Komagatella phaffii (Pichia pastoris). However, strong, fully constitutive genetic elements and expression cassettes for protein expression in Y. lipolytica remain limited. In this study, we leveraged genome-wide transcriptomics to uncover five strong promoters and four terminators. Among these, the promoter of ribosomal protein L41 demonstrated superior activity to the strongest previously reported promoters. We further demonstrated the functionality of pL41 across different media conditions and by using it to express diverse heterologous proteins. Similarly, we showed that the terminator of glutathione-S-transferase (tGST) supported higher protein expression and low transcriptional readthrough compared to commonly used terminators. To support protein secretion efforts, we utilized a secretomics-guided signal peptide screen to unveil three signal peptides, demonstrating broad applicability to different proteins. Integrating these genetic elements into a new expression cassette (YALI-pSTOmics1) resulted in a 3-fold increase in secretory expression of bovine fibroblast growth factor 2 compared to a combination of the best available state-of-the-art genetic tools for gene expression in Y. lipolytica. This expression cassette represents an open-source alternative to expensive commercial ones. Furthermore, the novel promoters and terminators provide options for metabolic engineering, where reuse of existing genetic parts is often a limitation.

Diabetes Mellitus is an epidemic affecting > 500 million, claiming 6-7 million lives annually. Chemically synthesised Glucagon-like peptide-1 receptor agonists (GLP-1RAs) containing artificial amino acids reduce haemoglobin A1c and obesity but are not yet affordable and require invasive injections. High dosage requirement and gastrointestinal complications are among the current limitations of oral GLP-1RAs. Therefore, we expressed codon optimised Exenatide and Lixisenatide fused with Cholera-toxin B-subunit (CTB) in lettuce chloroplasts to facilitate their oral delivery, increase affordability, and patient compliance. Site-specific integration of transgene expression cassettes into the chloroplast genome and removal of the selectable marker gene from marker-free lettuce transplastomic lines were confirmed using three sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. CTB-Exenatide and CTB-Lixisenatide expression levels were 1.94 and 3.64 mg/g plant powder in T0 generation and increased ~31 and ~48%, respectively in marker-removed T1 lines. Maternal inheritance of transgenes was confirmed by lack of segregation when seedlings were germinated in the selection medium before removal of the antibiotic resistance gene (aadA). Monosialotetrahexosylganglioside (GM1) ELISA confirmed pentameric assembly efficiency of both CTB-fusion proteins similar to commercial CTB standards. GLP-1 receptor binding confirmed functionality of CTB-Exenatide/CTB-Lixisenatide with statistical significance (***p < 0.001 by t-test) and post-translational amidation in chloroplasts. Expression of functional CTB-Exenatide and CTB-Lixisenatide in an edible marker-free system for the first time and much lower dosage requirement for functionality than recently developed synthetic GLP-1RAs paves the way for clinical studies to advance oral delivery of these affordable biologics.

Emerging and re-emerging swine viral infectious diseases impose substantial economic burdens. Additionally, swine, which frequently interact with humans, may facilitate virus evolution, posing a risk to public health security. Consequently, there is a pressing need to develop safe, effective, and rapid vaccine platforms, with vector vaccine being a viable option. In this study, we utilized the enhanced green fluorescent protein (eGFP) as an exogenous reporter to investigate the intergenic regions of the PIV5-JS17 strain for expressing exogenous proteins. These regions included N-P, P-M, M-F, F-SH, SH-HN, and HN-L. Our findings revealed that exogenous gene expression varied at different positions, with the expression cassette containing the non-coding sequence within the P-M intergenic region achieving the highest eGFP fluorescence intensity. Then, we successfully established a porcine infection model through oral administration of the recombinant virus, identified the target organs infected, and verified the safety of the viral vector in swine. Furthermore, using porcine deltacoronavirus (PDCoV)-S protein as a model antigen, it was demonstrated that the recombinant virus triggered a robust humoral and cellular immune responses. In conclusion, we have developed a novel oral gene delivery system for swine, providing insights and guidance for the design of vector vaccines based on the newly discovered porcine PIV5, the selection of appropriate exogenous gene insertion sites, and vaccine delivery strategies.IMPORTANCEThe research presented in this paper hinges on the fortunate isolation of the PIV5-JS17 strain from the intestines of pigs. Given the pressing demand for oral vaccines, the emergence of this novel PIV5 strain capable of infecting both the respiratory and intestinal tracts has sparked our interest in developing it as a vector vaccine. Utilizing eGFP as a model exogenous gene, our findings reveal that the P/M intergenic region serves as the optimal site for the insertion of exogenous genes. Using the PDCoV-S protein as a model antigen, the study shows that this novel porcine-derived PIV5 virus vector presents innovative prevention methods and gene delivery strategies for addressing porcine infectious diseases.

Secretory expression and fermentation optimization of recombinant porcine epidermal growth factor in Saccharomyces cerevisiae.

The transfer of apomixis, a clonal mode of reproduction by seeds, to crops has the potential to revolutionize agriculture by enabling the generation of one-line F1 hybrids that propagate clonally by seeds from one generation to the next. However, despite nearly four decades of intensive research, all the attempts to transfer the identified genetic determinants of apomixis from naturally apomictic wild plants to their crop relatives have failed to produce apomictic crops. Engineering of apomixis, mimicking the key features of a natural form of apomixis known as gametophytic diplosporous apomixis, has recently been achieved in rice and further improvements by the introduction of a single "all-in-one" T-DNA construct into calli derived from F1 hybrid seed embryos resulted in high frequency of clonal seeds. The T-DNA encodes a constitutively expressed Cas9 protein guided by sgRNAs designed to knock-out the functions of three genes essential for regulating crucial steps involved in meiosis, thereby eliminating meiosis and creating the Mitosis instead of Meiosis (MiMe) triple mutant. Additionally, the T-DNA contains another gene expression cassette consisting of a parthenogenetic trigger, the BABY BOOM1 (BBM1) transcription factor driven by an egg cell-specific (ECS) promoter. Inactivation of the three MiMe genes converts meiosis into mitosis-apomeiosis-yielding unrecombined and unreduced male and female spores developing into gametophytes. The BBM1 expression triggers parthenogenetic development of an embryo from the diploid egg cell of the female gametophyte. The endosperm develops sexually by the fusion of a diploid sperm cell and the central cell of the female gametophyte, which contains two diploid polar nuclei, resulting in an initially hexaploid endosperm. To date, the "all-in-one" T-DNA method has proved to be the most efficient for achieving high frequency (95-100% clonal seeds) synthetic apomixis in rice. Since the original publication, we have successfully generated synthetic apomictic events in three additional F1 hybrids. Here, we describe the methods for designing T-DNA constructs, analyzing mutations in first-generation (T0) MiMe mutant plants of F1 hybrids, and ascertaining the apomictic nature of the progenies from confirmed MiMe F1 hybrids, which relies on the egg cell-specific accumulation of BBM1.

Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells.

Reusable target-site toolkit for large-fragment (56.2 kilobases) chromosomal integration to enhance erythromycin biosynthesis in Escherichia coli.

Background and purpose:The estrogen receptor alpha-36 (ER-α36) is an alternative splice variant of classical ER-α66 and is abundantly present in both ER-α66-positive and ER-α66-negative breast tumor cells. Given its clinical relevance, developing targeted strategies against this isoform is of particular significance to breast cancer research. This study aimed to develop an ER-α36-specific recombinant biosimilar single-chain variable fragment (scFv) antibody.Experimental approach:The primary amino acid sequence of the anti-ER-α36 scFv was retrieved from patent US20110311517A1. An expression cassette harboring the scFv coding sequence was designed and incorporated into the backbone of the pET-28a(+) expression vector for recombinant expression in Escherichia coli (E. coli) BL21(DE3) cells. Expression conditions were then optimized, and the protein was purified using immobilized metal affinity chromatography. The binding of the purified scFv to ER-α36-expressing breast cancer cells was assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry.Findings/Results:Characterization using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting experiments revealed a molecular weight of 29 kDa for the expressed scFv antibody. Relative quantification revealed the highest scFv protein expression level 16 h after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside at 25 °C. Flow cytometry and ELISA assays demonstrated specific binding of the scFv to ER-α36 protein on MDA-MB-231 breast cancer cells, while no interaction was detected with ER-α36-negative MCF-10A normal mammary epithelial cell line.Conclusion/implications:The anti-ER-α36 scFv antibody fragment was successfully expressed using the E. coli expression system, and the purified protein was able to specifically recognize and bind to ER-α36-expressing human breast cancer cells.

Alternative AAV gene therapy for hemophilia A using expression of Bi8, a novel single-chain FVIII-mimetic antibody.

Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed. In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T1 generation on. In addition, the 22kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T0 plants targeting ZmSnRK2.1, to 87.5% and 100% for T1 plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T1 generation and 7 transgene-free homozygous snrk2.1 mutants in the T2 generation. The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.

Common variants of the apolipoprotein E (APOE) gene have a major impact on the risk of developing Alzheimer's disease (AD). Relative to homozygotes with the common E3 allele, the APOE4 variant (C112R) increases risk by 3.5-fold in E3/E4 heterozygotes and 15-fold in E4 homozygotes. Since the E3 and E4 alleles differ only by a single nucleotide, gene editing of E4 to E3 is a potential strategy to reduce AD risk in E4 homozygotes. Because the APOE pool in the brain is separate from systemic APOE, editing to treat AD would ideally be directed to the brain. Following in vitro optimization of prime editing guide RNAs, efficient prime editing expression cassettes were inserted into the adeno-associated virus (AAV) split-intein system and packaged into pairs of AAV vectors for invivo editing. The AAV vectors were administered to human homozygous APOE4-targeted replacement mice (TRE4), and APOE4 to APOE3 editing efficiency was assessed after 4 weeks. The prime editing construct designated APOE3/4-3_10 was the most efficient at APOE4 to APOE3 conversion, both in liver following intravenous delivery and in brain following intrahippocampal delivery. To assess brain-wide editing, two AAV capsids were compared, including AAVrh.10 with administration either directly to the hippocampus or to the cerebrospinal fluid via the cisterna magna and AAV-CAP.B10 administered intravenously. Other than minor differences in APOE4/3-3_10 mediated E4 to E3 editing in the cerebellum, the different capsids and routes yielded similar editing efficacy throughout the brain. This may represent a candidate treatment to reduce the risk of AD.

Arenin is a cystine-rich Kunitz-type protease inhibitor originally isolated from the skin secretion of the tree frog Dryophytes arenicolor, whose limited natural yield has hindered comprehensive functional studies. In this work, we established an efficient recombinant production system in Escherichia coli and evaluated its anticancer and wound-healing properties. A codon-optimized arenin gene, fused to an N-terminal 6 × His-TEV tag, was cloned into a T7-lac expression cassette. IPTG induction at 30 °C and 37 °C revealed distinct temperature-dependent partitioning: at 30 °C, arenin predominantly accumulated in the soluble fraction, whereas at 37 °C, it was confined to inclusion bodies. Both strategies yielded ≥ 90% pure peptide, producing 7.8 ± 0.6 mg and 12.4 ± 1.0 mg per 250 mL culture, respectively. Bioassays showed that HDFa fibroblasts tolerated 31.25–500 µg mL−1 arenin and had increased viability at 1000 µg mL−1. ER⁺ MCF-7 cells showed mild inhibition at low doses but growth stimulation at the highest, suggesting hormetic protease–receptor effects. Caco-2 cells were sensitive, with viability at 60.2 ± 3.4% at 31.25 µg mL−1 and below 80% up to 250 µg mL−1. Scratch-wound assays under serum deprivation or high glucose showed complete closure within 72 h at all arenin concentrations, comparable to Centella asiatica (10 µg mL−1), consistent with other Kunitz-family peptide activities in diverse protease environments. Overall, our results demonstrate the feasibility of a heterologous expression strategy for large-scale arenin production and highlight its dual functional potential, pro-regenerative effects in fibroblasts, and selective cytotoxicity toward hormone-independent cancer cells.Graphical abstract

Mucopolysaccharidosis VI (MPS VI) is a rare, autosomal recessive lysosomal storage disease caused by mutations in the arylsulfatase B gene (ARSB). Ocular manifestations of MPS VI include progressive corneal clouding, leading to vision loss. Herein, an adeno-associated virus (AAV) ARSB corneal gene addition strategy was evaluated in a naturally occurring MPS VI feline model. The AAV serotype 8 capsid was packaged with a single-strand optimized human ARSB expression cassette (optARSB) and administered to MPS VI feline corneas at a dose of 1e9 vector genomes via intrastromal injection. All AAV8-optARSB injections were well tolerated, resulting in a complete reversal of pre-existing corneal clouding within 2–3 weeks, which was maintained throughout the study. Sequential dosing of the contralateral cornea 7 weeks after the first dose also cleared the storage disease with similar kinetics despite more advanced disease. Confocal microscopy, histological analyses, and electron microscopy revealed disorganization in the posterior corneal stroma in untreated animals with AAV8-optARSB-treated corneas demonstrating improved morphology and tissue organization. Human arylsulfatase B was observed throughout the corneal stroma with decreased smooth muscle actin staining following AAV8-optARSB treatment. The collective results demonstrate that reversing feline MPS VI corneal clouding using intrastromal low-dose AAV8-optARSB is safe and effective. Furthermore, as this strategy relied on the same AAV capsid, vector dose, genetic cassette context, injection type, and volume deemed safe and effective for the treatment of MPS I, the data derived herein support a standardized pipeline for AAV corneal gene therapy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the selective deterioration of motor neurons in the central nervous system (CNS). A key driver of this pathogenesis is nuclear loss of ALS-associated protein TDP-43, leading to mis-splicing of TDP-43 targets including important neuronal genesSTMN2andUNC13A. Here, we have developed a gene therapy strategy for ALS and related TDP-43 proteinopathies, to correct mis-splicing of bothSTMN2andUNC13Acryptic exons using small nuclear RNAs (snRNAs) encoded from a single vector. We identified promoter sequence elements to increase therapeutic snRNA expression by 10-fold, then further optimized the expression cassette with combinatorial snRNA targeting to rescue multiple cryptic splicing targets. The engineered snRNAs restored normal pre-mRNA processing of bothSTMN2andUNC13Atranscripts despite TDP-43 loss of function, rescuing stathmin-2 protein levels in iPSC derived motor neurons, restoring their axonal regeneration capacity to wild-type levels. In addition, adeno-associated virus (AAV) delivery of the snRNAs to the murine central nervous system in the constitutive cryptic splicing modelStmn2HumΔGUfully restored corticalStmn2pre-mRNA processing, highlighting the utility of snRNAs as a therapeutic modalityin vivo. Together, this study demonstrates that snRNAs are a promising and versatile therapeutic strategy for the simultaneous correction of multiple aberrant transcripts affected by cryptic splicing in TDP-43 proteinopathies.

Ferritin-based hybrid protein particle vaccine combining M2e antigen of influenza A virus and S-protein tandem epitopes of SARS-CoV-2.

The yeast Cyberlindnera jadinii has long been used in food and feed industries. While it shows strong intracellular protein synthesis, its limited secretion capacity hampers its use as a protein production host. To overcome this, we used the endogenous invertase CjINV1 as a model and applied multidimensional engineering to the tetraploid wild-type strain NBRC0988. This involved multicopy integration of gene expression cassettes into the chromosome via a landing pad strategy, transcriptional enhancement through combinatorial optimization of promoter and terminator elements, and chitin-based cell wall remodeling to facilitate protein release. The engineered strain showed markedly enhanced protein secretion in a 5-L bioreactor, yielding 3456 ± 81 mg/L extracellular protein after 168 h cultivation, with invertase specific activity of 789.3 ± 52.3 U/mg. To our knowledge, this represents the highest reported yield for yeast-based invertase production to date, highlighting C. jadinii as a promising platform for recombinant protein production.

Streamlined construction of episomal vectors for rapid assessment of fusion phytase display in Saccharomyces cerevisiae.