A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

mRNA-1273 vaccinated inflammatory bowel disease patients receiving TNF inhibitors develop broad and robust SARS-CoV-2-specific CD8+ T cell responses.

SARS-CoV-2-specific CD8+ T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8+ T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8+ T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8+ T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8+ T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8+ T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8+ T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8+ T cells persisted and spike-specific CD8+ T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8+ T cell response.

Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration.

Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.

Parallel detection of SARS-CoV-2 epitopes reveals dynamic immunodominance profiles of CD8+ T memory cells in convalescent COVID-19 donors.

Objectives: High-magnitude CD8+ T cell responses are associated with mild COVID-19 disease; however, the underlying characteristics that define CD8+ T cell-mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope-specific CD8+ T cells remain largely unexplored and are essential for the development of next-generation broad-protective vaccines. This study identified a broad spectrum of conserved SARS-CoV-2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS-CoV-2 infection. Methods: CD8+ T cells from 51 convalescent COVID-19 donors were analysed for their ability to recognise 133 predicted and previously described SARS-CoV-2-derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in-depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels. Results: A comprehensive panel of 49 mostly conserved SARS-CoV-2-specific CD8+ T cell epitopes, including five newly identified low-magnitude epitopes, was established. We confirmed the immunodominance of HLA-A*01:01/ORF1ab1637-1646 and B*07:02/N105-113 and identified B*35:01/N325-333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361-369 and A*02:01/S269-277, depended on the donors' HLA-I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID-19 donors. Conclusion: SARS-CoV-2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS-CoV-2 epitopes, which likely contributes to long-term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross-reactive immune response, which could aid future vaccine strategies.

XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair.

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair.

Bulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. We find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

ERCC1 mutations impede DNA damage repair and cause liver and kidney dysfunction in patients.

ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.

BMP-2 gene delivery in cell-loaded and cell-free constructs for bone regeneration.

To induce osteogenicity in bone graft substitutes, plasmid-based expression of BMP-2 (pBMP-2) has been successfully applied in gene activated matrices based on alginate polymer constructs. Here, we investigated whether cell seeding is necessary for non-viral BMP-2 gene expression in vivo. Furthermore, to gain insight in the role of BMP-producing cells, we compared inclusion of bone progenitor cells with non-osteogenic target cells in gene delivery constructs. Plasmid DNA encoding GFP (pGFP) was used to trace transfection of host tissue cells and seeded cells in a rat model. Transgene expression was followed in both cell-free alginate-ceramic constructs as well as constructs seeded with syngeneic fibroblasts or multipotent mesenchymal stromal cells (MSCs). Titration of pGFP revealed that the highest pGFP dose resulted in frequent presence of positive host cells in the constructs. Both cell-loaded groups were associated with transgene expression, most effectively in the MSC-loaded constructs. Subsequently, we investigated effectiveness of cell-free and cell-loaded alginate-ceramic constructs with pBMP-2 to induce bone formation. Local BMP-2 production was found in all groups containing BMP-2 plasmid DNA, and was most pronounced in the groups with MSCs transfected with high concentration pBMP-2. Bone formation was only apparent in the recombinant protein BMP-2 group. In conclusion, we show that non-viral gene delivery of BMP-2 is a potentially effective way to induce transgene expression in vivo, both in cell-seeded as well as cell-free conditions. However, alginate-based gene delivery of BMP-2 to host cells or seeded cells did not result in protein levels adequate for bone formation in this setting, calling for more reliable scaffold compatible transfection methods.

Osteoinduction by Ex Vivo Nonviral Bone Morphogenetic Protein Gene Delivery Is Independent of Cell Type.

Ex vivo nonviral gene delivery of bone inductive factors has the potential to heal bone defects. Due to their inherent role in new bone formation, multipotent stromal cells (MSCs) have been studied as the primary target cell for gene delivery in a preclinical setting. The relative contribution of autocrine and paracrine mechanisms, and the need of osteogenic cells, remains unclear. This study investigates the contribution of MSCs as producer of transgenic bone morphogenetic proteins (BMPs) and to what extent the seeded MSCs participate in actual osteogenesis. Rat-derived MSCs or fibroblasts (FBs) were cotransfected with pBMP-2 and pBMP-6 or pBMP-7 via nucleofection. The bioactivity of BMP products was shown through in vitro osteogenic differentiation assays. To investigate their role in new bone formation, transfected cells were seeded on ceramic scaffolds and implanted subcutaneously in rats. Bone formation was assessed by histomorphometry after 8 weeks. As a proof of principle, we also investigated the suitability of bone marrow-derived mononuclear cells and the stromal vascular fraction isolated from adipose tissue for a one-stage gene delivery strategy. Bone formation was induced in all conditions containing cells overexpressing BMP heterodimers. Constructs seeded with FBs transfected with BMP-2/6 and MSCs transfected with BMP-2/6 showed comparable bone volumes, both significantly higher than controls. Single-stage gene delivery proved possible and resulted in some bone formation. We conclude that bone formation as a result of ex vivo BMP gene delivery can be achieved even without direct osteogenic potential of the transfected cell type, suggesting that transfected cells mainly function as a production facility for osteoinductive proteins. In addition, single-stage transfection and reimplantation of cells appeared feasible, thus facilitating future clinical translation of the method.

A novel injectable thermoresponsive and cytocompatible gel of poly(N-isopropylacrylamide) with layered double hydroxides facilitates siRNA delivery into chondrocytes in 3D culture.

Hybrid hydrogels composed of poly(N-isopropylacrylamide) (pNIPAAM) and layered double hydroxides (LDHs) are presented in this study as novel injectable and thermoresponsive materials for siRNA delivery, which could specifically target several negative regulators of tissue homeostasis in cartilaginous tissues. Effectiveness of siRNA transfection using pNIPAAM formulated with either MgAl-LDH or MgFe-LDH platelets was investigated using osteoarthritic chondrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing. No significant adverse effects of pNIPAAM/LDH hydrogels on cell viability were noticed. Cellular uptake of fluorescently labeled siRNA was greatly enhanced (>75%) in pNIPAAM/LDH hydrogel constructs compared to alginate, hyaluronan and fibrin gels, and was absent in pNIPAAM hydrogel without LDH platelets. When using siRNA against GAPDH, 82-98% reduction of gene expression was found in both types of pNIPAAM/LDH hydrogel constructs after 6 days of culturing. In the pNIPAAM/MgAl-LDH hybrid hydrogel, 80-95% of GAPDH enzyme activity was reduced in parallel with gene. Our findings show that the combination of a cytocompatible hydrogel and therapeutic RNA oligonucleotides is feasible. Thus it might hold promise in treating degeneration of cartilaginous tissues by providing supporting scaffolds for cells and interference with locally produced degenerative factors.

Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells.

The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.

Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate.

Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 degrees C; no pili were observed on cells grown at 21 degrees C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.