Interferon gene therapy with nadofaragene firadenovec for bladder cancer: from bench to approval.

Bladder cancer is a prevalent malignancy with limited therapeutic options, particularly for patients who are unresponsive to Bacillus Calmette-Guérin (BCG). The approval of interferon-α (IFNα) gene therapy with nadofaragene firadenovec (Adstiladrin®), the first gene therapy for genitourinary malignancies, has provided a promising alternative. This article reviews the research and milestones that led to the development and approval of nadofaragene firadenovec. Bladder cancer is well-suited for gene therapy due to direct access to the bladder and the availability of urine and tissue samples for monitoring. Early challenges included effective gene transfer across the urothelium, which was overcome initially by modulating the expression of coxsackie/adenovirus receptor (CAR) and, ultimately, by disrupting the urothelial barrier with Syn3. Nadofaragene firadenovec is a modified adenoviral vector carrying the IFNα gene. Clinical trials have shown promising results, with high response rates and manageable adverse events. Ongoing research focuses on improving patient selection, identifying biomarkers for response prediction, exploring alternative vectors for enhanced transfection efficiency, and developing combination strategies targeting resistance mechanisms. The approval of nadofaragene firadenovec marks a significant milestone in the field of gene therapy for bladder cancer, and future developments hold promise for further enhancing its efficacy and impact.

The current status of gene therapy in bladder cancer.

INTRODUCTION: Gene therapy aims to alter the biological properties of cells through the therapeutic delivery of nucleotides to treat a disease. Although originally developed to treat genetic disorders, the majority of gene therapy development today is for the treatment of cancer, including bladder cancer. AREAS COVERED: Following a brief history and a discussion of the mechanisms of gene therapy, we will focus on the current and future gene therapy strategies for bladder cancer. We will review the most consequential clinical trials published in the field. EXPERT OPINION: Recent transformative breakthroughs in bladder cancer research have deeply characterized the major epigenetic and genetic alterations of bladder cancer and have radically transformed our view of tumor biology and generated new hypotheses for therapy. These advances provided the opportunity to begin to optimize strategies for effective gene therapy for bladder cancer. Clinical trials have shown promising results, especially in BCG-unresponsive non-muscle-invasive bladder cancer (NMIBC), where effective second-line therapy remains an unmet need for patients facing cystectomy. Efforts are underway to develop effective combination strategies targeting resistance mechanisms to gene therapy for NMIBC.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Antiadenovirus Antibodies Predict Response Durability to Nadofaragene Firadenovec Therapy in BCG-unresponsive Non-muscle-invasive Bladder Cancer: Secondary Analysis of a Phase 3 Clinical Trial.

A recent phase 3 trial of intravesical nadofaragene firadenovec reported a promising complete response rate for patients with bacillus Calmette-Guérin-unresponsive non-muscle-invasive bladder cancer. This study examined the ability of antiadenovirus antibody levels to predict the durability of therapeutic response to nadofaragene firadenovec. A standardized and validated quantitative assay was used to prospectively assess baseline and post-treatment serum antibody levels among 91 patients from the phase 3 trial, of whom 47 (52%) were high-grade recurrence free at 12 mo (responders). While baseline titers did not predict treatment response, 3-mo titer >800 was associated with a higher likelihood of durable response (p = 0.026). Peak post-treatment titers >800 were noted in 42 (89%) responders versus 26 (59%) nonresponders (p = 0.001; assay sensitivity, 89%; negative predictive value, 78%). Moreover, 22 (47%) responders compared with eight (18%) nonresponders had a combination of peak post-treatment titers >800 and peak antibody fold change >8 (p = 0.004; assay specificity, 82%; positive predictive value, 73%). A majority of responders continued to have post-treatment antibody titers >800 after the first 6 mo of therapy. In conclusion, serum antiadenovirus antibody quantification may serve as a novel predictive marker for nadofaragene firadenovec response durability. Future studies will focus on large-scale validation and clinical utility of the assay. PATIENT SUMMARY: This study reports on a planned secondary analysis of a phase 3 multicenter clinical trial that established the benefit of nadofaragene firadenovec, a novel intravesical gene therapeutic, for the treatment of patients with bacillus Calmette-Guérin (BCG)-unresponsive high-risk non-muscle-invasive bladder cancer. Prospective assessment of serum anti-human adenovirus type-5 antibody levels of patients in this trial indicated that a combination of post-treatment titers and fold change from baseline can predict treatment efficacy. While this merits additional validation, our findings suggest that serum antiadenovirus antibody levels can serve as an important predictive marker for the durability of therapeutic response to nadofaragene firadenovec.

TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer.

Little is known regarding the subclone evolution process in advanced bladder cancer, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic bladder cancer through mRNA expression profiling of RNA isolated from 33 primary bladder cancer and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary bladder cancer compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were conducted in bladder cancer cell lines with high and low expression of TCF21, respectively. We also performed GEP in bladder cancer cell lines following TCF21 overexpression. We identified 2,390 genes differentially expressed in primary bladder cancer and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared with LN metastasis. We validated this finding with qPCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination, and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal bladder cancer cell lines decreased their invasive and metastatic potential. IMPLICATIONS: TCF21 is differentially overexpressed in primary bladder cancer compared with matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

Generation of a novel mouse strain with conditional, cell-type specific, expression of DND1.

Dead-End 1 (DND1) encodes an RNA binding protein critical for viable primordial germ cells in vertebrates. When introduced into cancer cell lines, DND1 suppresses cell proliferation and enhances apoptosis. However, the molecular function of mammalian wild-type DND1 has mostly been studied in cell lines and not verified in the organism. To facilitate study of wild-type DND1 function in mammalian systems, we generated a novel transgenic mouse line, LSL-FM-DND1 flox/+ , which conditionally expresses genetically engineered, FLAG-tagged and myc-tagged DND1 in a cell type-specific manner. We report that FLAG-myc-DND1 is indeed expressed in specific tissues of the mouse when LSL-FM-DND1 flox/+ is combined with mouse strains expressing Cre-recombinase. LSL-FM-DND1 flox/+ mice are fertile with no overt health effects. We expressed FLAG-myc-DND1 in the pancreas and found that chronic, ectopic expression of FLAG-myc-DND1 led to increase in fasting glucose levels in older mice. Thus, this novel LSL-FM-DND1 flox/+ mouse strain will facilitate studies on the biological and molecular function of wild-type DND1.

Inhibition of urothelial carcinoma through targeted type I interferon-mediated immune activation.

Type I interferon (IFN-I) has potent anti-tumor effects against urothelial carcinoma (UC) and may be an alternative treatment option for patients who do not respond to Bacillus Calmette-Guerin. However, the mechanisms that mediate the IFN-I-stimulated immune responses against UC have yet to be elucidated. Herein, we evaluated the anti-tumor mechanisms of IFN-I in UC in human patients and in mice. Patient tumors from a Phase I clinical trial with adenoviral interferon-α (Ad-IFNα/Syn3) showed increased expression of T cell and checkpoint markers following treatment with Ad-IFNα/Syn3 by RNAseq and immunohistochemistry analysis in 25% of patients. In mice, peritumoral injections of poly(I:C) into MB49 UC tumors was used to incite an IFN-driven inflammatory response that significantly inhibited tumor growth. IFN-I engaged both innate and adaptive cells, seen in increased intratumoral CD8 T cells, NK cells, and CD11b+Ly6G+ cells, but tumor inhibition was not reliant on any one immune cell type. Nonetheless, poly(I:C)-mediated tumor regression and change in the myeloid cell landscape was dependent on IL-6. Mice were also treated with poly(I:C) in combination with anti-PD-1 monoclonal antibody (mAb) to assess for additional benefit to tumor growth and animal survival. When used in combination with anti-PD-1 mAb, IFN-I stimulation prolonged survival, coinciding with inhibition of angiogenesis and enriched gene signatures of metabolism, extracellular matrix organization, and MAPK/AKT signaling. Altogether, these findings suggest IFN-I's immune-driven antitumor response in UC is mediated by IL-6 and a collaboration of immune cells, and its use in combination with checkpoint blockade therapy can increase clinical benefit.

The development of interferon-based gene therapy for BCG unresponsive bladder cancer: from bench to bedside.

BACKGROUND AND PURPOSE: BCG unresponsive bladder cancer is an inherently resistant disease state for which the preferred treatment is radical cystectomy. To date, no effective intravesical therapies exist for patients who possess these resistant tumors. For this reason, many research groups are actively investigating/testing novel therapeutic agents to aid in bladder preservation for this patient population. This review article describes our 15-year experience developing and testing IFN-based gene therapy. METHODS: A comprehensive review was performed of all studies pertaining to IFN-based gene therapy for non-muscle invasive bladder cancer from 2003 to 2018. RESULTS AND CONCLUSIONS: Over the past two decades, gene therapy has evolved into a powerful tool in our fight against cancer. After overcoming the initial barriers associated with gene delivery to the bladder, we have made significant strides forward in developing this novel therapeutic strategy for the treatment of this inherently resistant disease state. Our results to date are very encouraging; however, much work lies ahead to better understand and optimize this novel approach for treating non-muscle invasive bladder.

Effects of thiazolidinedione in patients with active bladder cancer.

OBJECTIVE: To examine the influence of perioperative thiazolidinedione (TZD) on cancer-specific outcomes in patients with diabetes mellitus (DM) undergoing radical cystectomy (RC) for urothelial carcinoma (UC). PATIENTS AND METHODS: A retrospective cohort of 173 patients with DM undergoing RC from 2005 to 2010 was identified. Of those, 53 were on TZD treatment at the time of RC, with 33 patients taking pioglitazone. Baseline clinicopathological characteristics, as well as cancer-specific survival (CSS), recurrence-free survival (RFS), and overall survival (OS) were compared between the patients on and off TZD therapy at the time of RC. In subgroup analysis, outcomes in patients specifically taking pioglitazone at the time of RC were compared to those not on a TZD. RESULTS: Baseline clinicopathological characteristics were similar between patients on and off TZD therapy at the time of RC. Overall, the median CSS rate was not reached in either group (P = 0.7). The estimated 5-year CSS was 67.8% in the non-TZD group and 66.3% in the TZD group. On multivariate analysis incorporating patient age, pathological T-staging, and adjuvant chemotherapy, TZD use was found not to be a significant predictor for CSS (hazard ratio 1.20, 95% confidence interval 0.66-2.17; P = 0.5). Additionally, RFS (P= 0.3) and OS (P = 0.2) were also similar between the two groups without adjusting for other variables. Comparison between patients taking pioglitazone vs patients not taking TZD yielded similar CSS (P = 0.2), RFS (P = 0.5), and OS (P= 0.2). CONCLUSIONS: CSS, as well as RFS and OS after RC were not compromised in patients on TZD therapy at the time of RC. Additional investigation is warranted in patients with non-muscle-invasive bladder cancer and muscle-invasive bladder cancer undergoing bladder-sparing procedures to assess the safety of using TZD in the setting of active UC.

Pim kinase isoforms: devils defending cancer cells from therapeutic and immune attacks.

Pim kinases are being implicated in oncogenic process in various human cancers. Pim kinases primarily deal with three broad categories of functions such as tumorigenesis, protecting cells from apoptotic signals and evading immune attacks. Here in this review, we discuss the regulation of Pim kinases and their expression, and how these kinases defend cancer cells from therapeutic and immune attacks with special emphasis on how Pim kinases maintain their own expression during apoptosis and cellular transformation, defend mitochondria during apoptosis, defend cancer cells from immune attack, defend cancer cells from therapeutic attack, choose localization, self-regulation, activation of oncogenic transcription, metabolic regulation and so on. In addition, we also discuss how Pim kinases contribute to tumorigenesis by regulating cellular transformation and glycolysis to reinforce the importance of Pim kinases in cancer and cancer stem cells.

Nephron Progenitor But Not Stromal Progenitor Cells Give Rise to Wilms Tumors in Mouse Models with ß-Catenin Activation or Wt1 Ablation and Igf2 Upregulation.

Wilms tumor, a common childhood tumor of the kidney, is thought to arise from undifferentiated renal mesenchyme. Variable tumor histology and the identification of tumor subsets displaying different gene expression profiles suggest that tumors may arise at different stages of mesenchyme differentiation and that this ontogenic variability impacts tumor pathology, biology, and clinical outcome. To test the tumorigenic potential of different cell types in the developing kidney, we used kidney progenitor-specific Cre recombinase alleles to introduce Wt1 and Ctnnb1 mutations, two alterations observed in Wilms tumor, into embryonic mouse kidney, with and without biallelic Igf2 expression, another alteration that is observed in a majority of tumors. Use of a Cre allele that targets nephron progenitors to introduce a Ctnnb1 mutation that stabilizes ß-catenin resulted in the development of tumors with a predominant epithelial histology and a gene expression profile in which genes characteristic of early renal mesenchyme were not expressed. Nephron progenitors with Wt1 ablation and Igf2 biallelic expression were also tumorigenic but displayed a more triphasic histology and expressed early metanephric mesenchyme genes. In contrast, the targeting of these genetic alterations to stromal progenitors did not result in tumors. These data demonstrate that committed nephron progenitors can give rise to Wilms tumors and that committed stromal progenitors are less tumorigenic, suggesting that human Wilms tumors that display a predominantly stromal histology arise from mesenchyme before commitment to a stromal lineage.

ß-catenin activation in a novel liver progenitor cell type is sufficient to cause hepatocellular carcinoma and hepatoblastoma.

Hepatocellular carcinoma (HCC) was thought historically to arise from hepatocytes, but gene expression studies have suggested that it can also arise from fetal progenitor cells or their adult progenitor progeny. Here, we report the identification of a unique population of fetal liver progenitor cells in mice that can serve as a cell of origin in HCC development. In the transgenic model used, mice carry the Cited1-CreER(TM)-GFP BAC transgene in which a tamoxifen-inducible Cre (CreER(TM)) and GFP are controlled by a 190-kb 5' genomic region of Cited1, a transcriptional coactivator protein for CBP/p300. Wnt signaling is critical for regulating self-renewal of progenitor/stem cells and has been implicated in the etiology of cancers of rapidly self-renewing tissues, so we hypothesized that Wnt pathway activation in CreER(TM)-GFP(+) progenitors would result in HCC. In livers from the mouse model, transgene-expressing cells represented 4% of liver cells at E11.5 when other markers were expressed, characteristic of the hepatic stem/progenitor cells that give rise to adult hepatocytes, cholangiocytes, and SOX9(+) periductal cells. By 26 weeks of age, more than 90% of Cited1-CreER(TM)-GFP;Ctnnb1(ex3(fl)) mice with Wnt pathway activation developed HCC and, in some cases, hepatoblastomas and lung metastases. HCC and hepatoblastomas resembled their human counterparts histologically, showing activation of Wnt, Ras/Raf/MAPK, and PI3K/AKT/mTOR pathways and expressing relevant stem/progenitor cell markers. Our results show that Wnt pathway activation is sufficient for malignant transformation of these unique liver progenitor cells, offering functional support for a fetal/adult progenitor origin of some human HCC. We believe this model may offer a valuable new tool to improve understanding of the cellular etiology and biology of HCC and hepatoblastomas and the development of improved therapeutics for these diseases.

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier.

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice.

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain.

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

Impaired wound healing in mice lacking the basement membrane protein nidogen 1.

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable alpha-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the beta1 and beta4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.

Basement membranes in skin are differently affected by lack of nidogen 1 and 2.

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.

Nidogens-Extracellular matrix linker molecules.

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.

Lack of nidogen-1 and -2 prevents basement membrane assembly in skin-organotypic coculture.

Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Furthermore, in various locations, extravasation of erythrocytes was observed implying microvascular defects. Mice expressing solely either isoform, had a functional BM, although nidogen-2 binds with lower affinity to the laminin gamma1 chain. Having previously blocked BM formation by interfering with nidogen-1 binding to laminin in skin-organotypic cocultures, here we investigated the roles of nidogen-1 and -2 in this model. For that purpose, human HaCaT cells were grown in three-dimensional cocultures on collagen matrices containing murine fibroblasts of varying nidogen deficiency. As with our experiments blocking laminin-nidogen interaction, lack of both nidogens completely prevented BM deposition and ultrastructural assembly of BM and hemidesmosomes, although other BM proteins remained detectable at comparable levels with no signs of degradation. Supplementation by recombinant nidogen-1 or -2 restored these structures, as shown by immunofluorescence and electron microscopy, confirming that in this system nidogen-2 is equivalent to nidogen-1, and both can promote the development of a functional BM zone.

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice.

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.