Increased Transgene Expression Research Articles

Synergistic effects of herpes oncolytic virus and cyclophosphamide for recurrent malignant glioma: a narrative review

Gliomas, comprising nearly 80% of brain malignancies, present a formidable challenge with glioblastomas being the most aggressive subtype. Despite multidisciplinary care, including surgery and chemoradiotherapy, the prognosis remains grim, emphasizing the need for innovative treatment strategies. The blood-brain barrier complicates drug access, and the diverse histopathology hinders targeted therapies. Oncolytic herpes viruses (oHSVs), particularly HSV1716, G207, and rQNestin34.5v, show promise in glioma treatment by selectively replicating in tumor cells. Preclinical and clinical studies demonstrate the safety and efficacy of oHSVs, with T-Vec being FDA-approved. However, challenges like viral delivery limitations and antiviral responses persist. The combination of oHSVs and combining cyclophosphamide (CPA) addresses these challenges, demonstrating increased transgene expression and viral activity. The immunosuppressive properties of CPA, particularly in metronomic schedules, enhance oHSV efficacy, supporting the development of this combination for recurrent malignant gliomas. CPA with oHSVs enhances viral oncolysis and extends survival. CPA’s immunomodulatory effects, suppressing regulatory T cells, improve oHSV efficiency. While obstacles remain, this synergistic approach offers hope for improved outcomes, necessitating further research and clinical validation.

Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target.

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

Next-generation strategies to improve safety and efficacy of adeno-associated virus-based gene therapy for hemophilia: lessons from clinical trials in other gene therapies.

Three major directions for the global progress of adeno-associated virus (AAV) vectors for gene therapies (GT) are analyzed: a) engineering vectors to increase transgene expression; b) aligning interests of the health system with costs and challenges for pharmaceutical industry; c) refining patient eligibility criteria, and endpoints definition. Currently employed AAV vectors may cause toxicity and adverse events. Furthermore, studies in animals do not fully predict risks and clinical benefits of AAV-based GT, and animal models reflecting the heterogeneity of certain clinical settings (e.g., congestive heart failure) are poorly available for improving AAV-based GT. Finally, antisense and gene editing approaches will soon complement gene augmentation strategies for the stable solution of unsolved issues of AAV-based GT. While minimizing toxicity, next-generation AAV vectors should decrease the viral load needed to achieve therapeutic efficacy; be functional in a restricted cellular subset; avoid transgene expression in unwanted cells (e.g., hepatocytes), and escape immune oversight in AAV-based GT. The role of stress-induced apoptosis in the loss of transgene expression in GT should be also explored. Aligning interests and obligations of pharmaceutical industry with those of the health system is critical for AAV-based GT success. Costs and challenges for pharmaceutical industry include a) removing impurities from AAV; b) validating tests to measure treatment efficacy, c) promoting training programs to standardize vector genomes delivery, d) collecting long-term follow-up data, and e) maintaining sustainability and cost-effectiveness of AAV-based GT. In rare disorders with small patient numbers (e.g., hemophilia), clearcut outcomes are mandatory as endpoints of unequivocal efficacy data.

A C1qTNF3 collagen domain fusion chaperones diverse secreted proteins and anti-Aβ scFvs: Applications for gene therapies

Enhancing production of protein cargoes delivered by gene therapies can improve efficacy by reducing the amount of vector or simply increasing transgene expression levels. We explored the utility of a 126-amino acid collagen domain (CD) derived from the C1qTNF3 protein as a fusion partner to chaperone secreted proteins, extracellular "decoy receptor" domains, and single-chain variable fragments (scFvs). Fusions to the CD domain result in multimerization and enhanced levels of secretion of numerous fusion proteins while maintaining functionality. Efficient creation of bifunctional proteins using the CD domain is also demonstrated. Recombinant adeno-associated viral vector delivery of the CD with a signal peptide resulted in high-level expression with minimal biological impact as assessed by whole-brain transcriptomics. As a proof-of-concept invivo study, we evaluated three different anti-amyloid Aβ scFvs (anti-Aβ scFvs), alone or expressed as CD fusions, following viral delivery to neonatal CRND8 mice. The CD fusion increased half-life, expression levels, and improved efficacy for amyloid lowering of a weaker binding anti-Aβ scFv. These studies validate the potential utility of this small CD as a fusion partner for secretory cargoes delivered by gene therapy and demonstrate that it is feasible to use this CD fusion to create biotherapeutic molecules with enhanced avidity or bifunctionality.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese-hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MAR), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR-1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+ hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells. This article is protected by copyright. All rights reserved.

An optimized prime editing system for efficient modification of the pig genome.

Prime editing (PE) is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-to-base conversions. However, its low efficiency hampers the application in creating novel breeds and biomedical models, especially in pigs and other important farm animals. Here, we demonstrate that the pig genome is editable using the PE system, but the editing efficiency was quite low as expected. Therefore, we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts (PEFs). First, we modified the pegRNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine, which significantly enhanced PE efficiency by improving the expression of pegRNA and targeted cleavage. Then, we targeted SAMHD1, a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that impedes the reverse transcription process in retroviruses, and found that treatment with its inhibitor, cephalosporin C zinc salt (CPC), increased PE efficiency up to 29-fold (4-fold on average), presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) in the PE system. Moreover, PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors (HDACis). Among the four HDACis tested, panobinostat was the most efficient, with an efficiency up to 122-fold (7-fold on average), partly due to the considerable HDACi-mediated increase in transgene expression. In addition, the synergistic use of the three strategies further enhanced PE efficiency in PEFs. Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

Focused ultrasound enhances transgene expression of intranasal hGDNF DNA nanoparticles in the sonicated brain regions.

The therapeutic potential of many gene therapies is limited by their inability to cross the blood brain barrier (BBB). While intranasal administration of plasmid DNA nanoparticles (NPs) offers a non-invasive approach to bypass the BBB, it is not targeted to disease-relevant brain regions. Here, our goal was to determine whether focused ultrasound (FUS) can enrich intranasal delivery of our plasmid DNA NPs to target deeper brain regions, in this case the regions most affected in Parkinson's disease. Combining FUS with intranasal administration resulted in enhanced delivery of DNA NPs to the rodent brain, by recruitment and transfection of microglia. FUS increased transgene expression by over 3-fold after intranasal administration compared to intravenous administration. Additionally, FUS with intranasal delivery increased transgene expression in the sonicated hemisphere by over 80%, altered cellular transfection patterns at the sonication sites, and improved penetration of plasmid NPs into the brain parenchyma (with a 1-fold and 3-fold increase in proximity of transgene expression to neurons in the forebrain and midbrain respectively, and a 40% increase in proximity of transgene expression to dopaminergic neurons in the substantia nigra). These results provide evidence in support of using FUS to improve transgene expression after intranasal delivery of non-viral gene therapies.

Interplay between Furin and Sialoglycans in Modulating Adeno-Associated Viral Cell Entry.

Adeno-associated viruses (AAVs) are small, helper-dependent, single-stranded DNA viruses that exploit a broad spectrum of host factors for cell entry. During the course of infection, several AAV serotypes have been shown to transit through the trans-Golgi network within the host cell. In the current study, we investigated whether the Golgi-localized, calcium-dependent protease furin influences AAV transduction. While CRISPR/Cas9-mediated knockout (KO) of the Furin gene minimally affected the transduction efficiency of most recombinant AAV serotypes tested, we observed a striking increase in transgene expression (~2 log orders) for the African green monkey isolate AAV4. Interrogation of different steps in the infectious pathway revealed that AAV4 binding, uptake, and transcript levels are increased in furin KO cells, but postentry steps such as uncoating or nuclear entry remain unaffected. Recombinant furin does not cleave AAV4 capsid proteins nor alter cellular expression levels of essential factors such as AAVR or GPR108. Interestingly, fluorescent lectin screening revealed a marked increase in 2,3-O-linked sialoglycan staining on the surface and perinuclear space of furin KO cells. The essential nature of increased sialoglycan expression in furin KO cells in enhancing AAV4 transduction was further corroborated by (i) increased transduction by the closely related isolates AAVrh.32.33 and sea lion AAV and (ii) selective blockade or removal of cellular 2,3-O-linked sialoglycans by specific lectins or neuraminidase, respectively. Based on the overall findings, we postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn can influence permissivity to AAVs and possibly other viruses. IMPORTANCE Adeno-associated viruses (AAVs) are a proven recombinant vector platform for gene therapy and have demonstrated success in the clinic. Continuing to improve our knowledge of AAV-host cell interactions is critical for improving the safety and efficacy. The current study dissects the interplay between furin, a common intracellular protease, and certain cell surface sialoglycans that serve as viral attachment factors for cell entry. Based on the findings, we postulate that differential expression of furin in host cells and tissues is likely to influence gene expression by certain recombinant AAV serotypes.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

A novel hybrid promoter capable of continuously producing proteins in high yield

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level. Expression levels of transgenes by each promoter were measured at transient and stable levels for five single promoters: Rous sarcoma virus (RSV), cytomegalovirus (CMV), human phosphoglycerate kinase (hPGK), simian virus 40 (SV40), and zebrafish ubiquitin B (Ubb). The hPGK promoter enabled high-yield transgene expression at transient levels and the SV40 promoter enabled sustained expression at stable levels. Therefore, hPGK and SV40 promoters were selected as candidates for establishing hybrid promoters and two hybrid promoters were constructed; one hybrid promoter in which the SV40 promoter is added before the hPGK promoter (a.k.a. SKYI) and the other hybrid promoter in which the SV40 promoter is added after the hPGK promoter (a.k.a. SKYII). Of the two hybrid promoters, the hybrid promoter SKYII promoted high-yield transgene expression at both transient and stable levels compared to single hPGK and SV40. Together, our findings open new doors in the field of biopharmaceuticals by presenting a novel promoter platform that can be used for high-yield and sustained protein production.

Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells

Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies.

Enhancement of healthful novel sugar contents in genetically engineered sugarcane juice integrated with molecularly characterized ThSyGII (CEMB-SIG2)

Enhancement of sugar contents and yielding healthful sugar products from sugarcane demand high profile scientific strategies. Previous efforts to foster manipulation in metabolic pathways or triggering sugar production through combating abiotic stresses fail to yield high sugar recovery in Saccharum officinarum L. Novel sucrose isomers trehalulose (TH) and isomaltulose (IM) are naturally manufactured in microbial sources. In pursuance of novel scientific methodology, codon optimized sucrose isomerase gene, Trehalulose synthase gene II(CEMB-SIG2) cloned under dual combined stem specific constitutive promoters in pCAMBIA1301 expression vector integrated with Vacuole targeted signal peptide (VTS) to concentrate gene product into the vacuole. The resultant mRNA expression obtained by Real Time PCR validated extremely increased transgene expression in sugarcane culms than leaf tissues. Overall sugar estimation from transgenic sugarcane lines was executed through refractometer. HPLC based quantifications of Trehalulose (TH) alongside different internodes of transgenic sugarcane confirmed the enhancement of boosted sugar concentrations in mature sugarcane culms. Trehalulose synthase gene II receptive sugarcane lines indicated the unprecedented impressions of duly combined constitutive stem regulated promoters. Transgenic sugarcane lines produce highest sugar recovery percentages, 14.9% as compared to control lines (8.5%). The increased sugar recovery percentage in transgenic sugarcane validated the utmost performance and expression of ThSyGII gene .High Profile Liquid chromatography based sugar contents estimation of Trehalulose (TH) and Isomaltulose (IM) yielded unprecedented improvement in the whole sugar recovery percentage as compared to control lines.⁠

Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing.

An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5' or 3' processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.

Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability.

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

Stabilizing and Anti-Repressor Elements Effectively Increases Transgene Expression in Transfected CHO Cells.

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells.

The generation of the stable, high-level recombinant protein-producing cell lines remains a significant challenge in the biopharmaceutical industry. Expression vector optimization is an effective strategy to increase transgene expression levels and stability, and the choice of suitable poly A element is crucial for the expression of recombinant protein. In this study, we investigated the effects of different poly A elements on transgene expression in Chinese hamster ovary (CHO) cells. Five poly A elements, including bovine growth hormone (BGH), mutant BGH, herpes simplex virus type 1 thymidine kinase (HSV-TK), SV40, and a synthetic (Synt) poly A, were cloned into the expression vector and transfected into CHO cells. The results indicated the SV40 and Synt poly A sequences can significant improve eGFP transgene expression in stable transfected CHO cells and maintain long-term expression. However, qPCR results showed that the eGFP expression at protein level was not related to the gene copy number and mRNA level. Importantly, the SV40 and Synt poly A elements decreased the variation of eGFP transgene expression. Furthermore, it also showed that the SV40 and Synt poly A elements induced higher levels of adalimumab expression. In conclusion, SV40 poly A and Synt poly A are stronger elements that increase stable transgene expression and decrease the variation of expression, and the choice of suitable poly A element is helpful to improve the expression of recombinant protein.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5′-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5′-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

Blocking T-cell egress with FTY720 extends DNA vaccine expression but reduces immunogenicity.

Optimal immunogenicity from nucleic acid vaccines requires a balance of antigen expression that effectively engages the host immune system without generating a cellular response that rapidly destroys cells producing the antigen and thereby limiting vaccine antigen expression. We investigated the role of the cellular response on the expression and antigenicity of DNA vaccines using a plasmid DNA construct expressing luciferase. Repeated intramuscular administration led to diminished luciferase expression, suggesting a role for immune‐mediated clearance of expression. To investigate the role of cell trafficking, we used the sphingosine 1‐phosphate receptor (S1PR) modulator, FTY720 (Fingolimod), which traps lymphocytes within the lymphoid tissues. When lymphocyte trafficking was blocked with FTY720, DNA transgene expression was maintained at a constant level for a significantly extended time period. Both continuous and staggered administration of FTY720 prolonged transgene expression. However, blocking lymphocyte egress during primary transgene administration did not result in an increase of transgene expression during secondary administration. Interestingly, there was a disconnect between transgene expression and immunogenicity, as increasing expression by this approach did not enhance the overall immune response. Furthermore, when FTY720 was administered alongside a DNA vaccine expressing the HIV gp140 envelope antigen, there was a significant reduction in both antigen‐specific antibody and T‐cell responses. This indicates that the developing antigen‐specific cellular response clears DNA vaccine expression but requires access to the site of expression in order to develop an effective immune response.

Establishment of a second generation homozygous CRISPRa human induced pluripotent stem cell (hiPSC) line for enhanced levels of endogenous gene activation

CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.