Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes

Martínez-Balsalobre, Elena; García-Castillo, Jesús; García-Moreno, Diana; Naranjo-Sánchez, Elena; Fernández-Lajarín, Miriam; Blasco, María A.; Alcaraz-Pérez, Francisca; Mulero, Victoriano; Cayuela, María L.

doi:10.1038/s41467-023-41472-7

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Published: 22 September 2023

Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes

Nature Communications volume 14, Article number: 5912 (2023) Cite this article

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Abstract

Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.

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Introduction

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric repeats at the end of eukaryotic chromosomes to prevent telomeric shortening¹. This enzyme consists mainly of a protein component (TERT) and an RNA component (TERC)². TERC has a very complex structure characterized by the presence of three conserved structural domains: the pseudoknot/core domain, which contains the template sequence and is essential for telomerase activity, the conserved regions 4 and 5 (CR4/CR5), which are critical for TERT association, and the ScaRNA domain, which contains the H/ACA and CR7 domains that are responsible for interacting with other telomerase-associated proteins^3,4.

Telomerase mutations are associated with premature ageing, while telomerase reactivation in adult cells is associated with cancer⁵. However, numerous studies have shown that both components of telomerase complex are involved in several cell signaling pathways without any apparent involvement of its well established function in telomere maintenance^6,7. Specifically, our laboratory has discovered that TERC has an extracurricular role in the regulation of myelopoiesis in both zebrafish and humans^8,9. In these models, TERC behaves as a typical transcription factor that regulates myelopoiesis through regulating the expression of the master myeloid genes csf3b (colony-stimulating factor 3b) and spi1b (Spi1 proto-oncogene b) in zebrafish and CSF2 and SPI1 in humans by recruiting RNA polymerase II (RNA Pol II) to a consensus DNA binding sequence found in their promoters or enhancers¹⁰. Thus, terc overexpression promotes myelopoiesis, increasing the number of neutrophils and macrophages, while terc deficiency results in neutropenia and monocytopenia in zebrafish^8,9. Importantly, the CR4/CR5 domain of TERC is essential for performing this novel role in the regulation of myelopoiesis, since a mutation of this domain found in dyskeratosis congenita (DC) patients, but not patient mutations affecting other domains, results in the inability of TERC to properly regulate myelopoiesis and to physically interact with RNA Pol II, despite its DNA binding capacity being unaffected⁹. These results might pave the way for the generation of new tools for the treatment of pathologies in which neutropenia is a hallmark.

Neutropenia is a dangerous and potentially fatal disease characterized by a lower than normal number of neutrophils in the circulation, due to reduced production, accelerated elimination or storage problems. Neutropenia can be caused by multiple factors, such as chemotherapy, drugs, infections, autoimmune diseases, bone marrow disorders, chronic idiopathic neutropenia¹¹ or genetic disorder (as it happens in DC)^12,13. DC is an inherited sickness associated with mutations in telomerase components or in telomere-stabilizing components¹⁴. The principal cause of premature mortality is bone marrow failure, associated with the development of aplastic anemias and myelodysplastic syndromes^15,16. The incidence of these hematopoietic phenotypes is higher in patients harboring mutations that affect TERC than in those patients with other mutations, an observation that cannot be explained by telomere shortening alone¹⁷. It is tempting to speculate, therefore, that these hematopoietic syndromes are related with the role of TERC in myelopoiesis.

The currently available treatment for neutropenia is the administration of granulocyte colony stimulating factor (GCSF, encoded by the CSF3 gene)^11,18. GCSF has been approved by the Food and Drug Administration (FDA) for the treatment of congenital and acquired neutropenia and for the mobilization of peripheral hematopoietic progenitor cells for stem cell transplantation¹⁹. In addition, GCSF therapy has been used in DC patients, being relatively effective in children and young adults suffering this disorder^20,21,22,23. However, while GCSF treatment has some side effects, the main disadvantage is its high cost. Since increased TERC expression activates myelopoiesis, precisely through the induction of GCSF among other myeloid genes, this could be a strategy for treating neutropenia, monocytopenia or telomeric diseases, such as DC. In addition, RNA synthesis is cheaper compare with GCSF production. Unfortunately, it is not feasible to manipulate TERC expression in patients, as it is in zebrafish or cell line models. Therefore, we decided to design a series of RNA aptamers based on the CR4/CR5 domain of TERC trying to mimic TERC overexpression effect observed in our experimental models.

Aptamers are synthetic single-stranded DNA or RNA sequences that adopt unique three-dimensional structures that allow them to recognize a specific target with high affinity²⁴. They have been used as therapeutic agents in several diseases^25,26. In the present study, several aptamers based on the CR4/CR5 domain of human and zebrafish TERC are designed and characterized. The results demonstrate that they behave as endogenous TERC enforcing myelopoiesis through the recruitment of RNA Pol II to master myeloid genes. In addition, preclinical zebrafish models of DC and poikiloderma with neutropenia and iPSC human model point to their usefulness to treat congenital neutropenia caused by different genetic alterations.

Results

Aptamers were designed based on CR4/CR5 domain of TERC

We have previously shown that TERC plays an essential role in myelopoiesis by increasing myeloid gene expression^8,9. As the CR4/CR5 domain of TERC is responsible for this noncanonical function, it was decided to design several aptamers based on the zebrafish CR4/CR5 terc domain to test its potential clinical usefulness in blood disorders. The first aptamer was full CR4/CR5 domain, known as T800 aptamer, and the second one was T1000 aptamer, consisting of only the P6.1 fork of the CR4/CR5 domain. This fork was selected for study because the mutation G305A in humans (G194A in zebrafish) has been shown to produce aplastic anemia (AA)²⁷. As a control, another aptamer was designed from the P6.1 fork but containing the G194A mutation, leading to it being called T1000_mut aptamer (Figs. S1a–S1c). Notably, this mutation drastically altered the aptamer structure (Fig. S1d), suggesting the importance of the tertiary structure of terc to perform its noncanonical function in myelopoiesis and anticipating its relevance in DC. As an additional control, we design an aptamer based in the CR7 fork of ScaRNA domain, called CR7 aptamer, which is not involved in myelopoiesis, since the mutation of this domain found in DC patient was able to increase myelopoiesis as wild type terc did⁹ (Figs. S1a–S1d). None of the used aptamers affected embryo development (Table S1).

Mouse and human aptamers were also designed and they had similar structures to the zebrafish ones (Figs. S1c, S1d). In addition, the mutation of the control aptamers (T1000_mut) found in AA patients (G305A)²⁷ dramatically changed their structure (Figs. S1c, S1d). As an additional control, human CR7 aptamer was also designed. All the human and mouse aptamers were chemically modified on the sugar ring, replacing the 2′ position with an O-methyl group (OCH3) on the first and last two bases of every aptamer to increase stability and prevent degradation by nucleases^28,29,30. Furthermore, they were labeled at their 3′ end by Cy3 fluorescent dye to facilitate tracking.

Aptamers increase myelopoiesis, without affecting erythropoiesis, in zebrafish

To evaluate the effect of terc-derived aptamers on hematopoiesis, they were microinjected into zebrafish embryos at the one-cell stage, and the larvae were analyzed 48 h later (Fig. 1a). The levels of every aptamer were checked by RT-qPCR in every single experiment and if they were not detected, the experiment was discarded (Fig. S2a).

**Fig. 1: T800 and T1000 aptamers enforce myelopoiesis in zebrafish.**

T800 and T1000 aptamers were able to increase neutrophil and macrophage numbers at full terc level, assayed in mpx:GFP and mpeg1:GFP transgenic zebrafish lines, respectively (Fig. 1b, c). However, the CR7 aptamer failed to increase neutrophil and macrophage numbers, confirming the specificity of the observed effects with T800 and T1000 aptamers and supporting our hypothesis that individual domains of terc can perform its function in myelopoiesis in the same way as the full molecule. Importantly, the T1000_mut aptamer failed to affect the number of myeloid cells, demonstrating that this point mutation has a drastic effect on T1000 function, suggesting the importance of a correct tertiary structure for its function in the regulation of myelopoiesis and confirming the relevance of this mutation in DC pathology. Furthermore, there were no differences in the erythrocyte number (lcr:GFP positive cells) of larvae microinjected with different aptamers or full terc (Fig. 1d), confirming previous results obtained with full terc⁸. Together, these results indicate that aptamers promote myelopoiesis without affecting erythropoiesis, an observation that is crucial for their possible therapeutic use.

As terc regulates neutrophil numbers in zebrafish larvae through fine-tuning the expression levels of the main myeloid (spi1b) and erythroid (gata1a) transcription factors and csf3b mRNA expression⁸, the transcript levels of these genes were analyzed by RT-qPCR in larvae treated with the different aptamers. The results showed that although the T800 and T1000 aptamers were able to increase spi1b and csf3b transcript levels and the spi1b/gata1 ratio in the same way as full terc, the CR7 and T1000_mut negative controls failed to do so (Fig. S2b), which would explain the observed increase in neutrophil and macrophage numbers (Fig. 1b).

As aptamers may be recognized as foreign RNA by innate immune system, we checked the expression of several inflammatory and interferon gene markers, including interferon-stimulated gene 15 (isg15), NLR family pyrin domain containing 3 (nlrp3) and tumor necrosis factor α (tnfa). The results showed that the transcript levels of none of these genes were affected by the aptamers (Fig. S2c). In addition, neutrophil activity, assayed by a recruitment assay to wound, was also unaffected (Fig. 1e). Thus, although the neutrophil number at the injury site was higher at all time points analyzed in T1000 aptamer treated larvae compared with T1000_mut aptamer or without treatment, the kinetics of the recruitment was similar in both groups (Fig. 1e).

Aptamers physically bind to DNA and to RNA pol II

It has been shown that terc regulates the expression of myeloid genes by binding to consensus sequences¹⁰, called terc binding sites (tercbs), and by recruiting the transcriptional machinery⁹. As these sequences are found in the csf3b promoter, it was cloned into a luciferase reporter for in vivo transcription activity assays. Fig. 2a shows that, while the T800 and T1000 aptamers were both able to increase wild type csf3b promoter activity at similar levels to full terc, the promoter lacking the tercbs did not change upon treatment by terc or aptamers. As expected, the CR7 and T1000_mut aptamers were not able to increase the activity of either wild type or mutant csf3b promoters. Furthermore, either genetic deletion of tercbs of csf3b promoter (Fig. 2b) or Csf3a/Csf3b deficiency (Fig. 2c) impaired the induction of myelopoiesis by T1000 aptamer. This demonstrated that the tercbs region was necessary for csf3b expression regulation by aptamers, as occurs with terc, confirming that aptamers regulate myelopoiesis using the same mechanism as full terc.

**Fig. 2: T1000 aptamer enforce myelopoiesis by activating *gcsf3b tercbs* and *gcsfa/b* expression.**

As the simplest theory to explain the action mechanism of aptamers is an indirect effect through the modulation of endogenous terc, the RNA levels of terc were measured by RT-qPCR, and the results showed that its transcript levels were unaltered by aptamers (Fig. 3a). In was also thought interesting to know if aptamers affect telomerase activity, and, while it was found that neither aptamers nor full terc affected telomerase activity, the telomerase activity inhibitor BIBR1532 reduced it more than 50%, as was expected (Fig. 3b). Therefore, the ability of aptamers to promote myelopoiesis is not mediated through the regulation of endogenous terc or telomerase activity.

**Fig. 3: Aptamers physically bind to DNA and to RNApolII.**

The ability of aptamers to increase neutrophil and macrophage numbers through engaging the tercbs in the csf3b promoter led us to hypothesize that aptamers were acting as terc. To confirm the direct binding of aptamers to DNA, an aptamer-DNA binding assay was performed to evaluate whether aptamers were able to bind to putative tercbs in vitro. For this, a dsDNA biotinylated probe of csf3b upstream regulatory sequences (100 bp) containing tercbs was incubated with aptamers and terc, as positive control, or luciferase mRNA (luc), as negative control. In parallel, the same experiment was performed with a csf3b promoter probe with mutant tercbs. The results showed that terc was able to bind to the csf3b probe with very high affinity compared to the control probe (luc) (Fig. 3c), confirming the relevance of tercbs for terc binding to DNA⁹. Similarly, the T800 aptamer robustly bound to wild type csf3b probe compared with the rest of aptamers and full terc, while T1000 and T1000_mut interacted weakly with the probe, all interactions being tercbs-dependent (Fig. 3c). However, both human T800 and T1000 aptamers interacted in vivo with similar affinities with the TERCbs of CSF2 and SPI1 promoters in HL-60 cells (Fig. 3d). These results show that T800 and T1000 aptamers can directly bind the tercbs.

These results prompted us to check whether the aptamers were also able to interact with RNA Pol II, as could terc⁹. For this, an RNA pulldown procedure was performed using biotinylated aptamers incubated with protein extract from 5-day-old zebrafish larvae. Biotinylated terc, as positive control, and biotinylated GFP mRNA, as negative control, were also included. It was found that both T800 and T1000 aptamers interacted with RNA Pol II with similar efficiency to that of terc (Fig. 3e). As expected, the negative control aptamers CR7 and T1000_mut did not show a statistically significant interaction with RNA Pol II. Therefore, the aptamer action mechanism seems to be similar to that of terc, since aptamers are able to bind to DNA and to RNA pol II.

Aptamers rescue neutropenia in zebrafish disease models of dyskeratosis congenita and poikiloderma with neutropenia

TERC haploinsufficiency generates telomeropathies, such as DC, because telomerase is unable to maintain telomere length in tissues that need constant renewal, such as the hematopoietic system¹⁶. Therefore, it was decided to test aptamers in heterozygous terc (terc ^+/-) and homozygous (terc ^-/-) zebrafish lines, both of which show telomere shortening and neutropenia at 48 hpf (Fig. 4a). Notably, the T800 and T1000 aptamers were able to rescue neutropenia of both lines, in the same way as terc (Fig. 4a). This result is of highly relevance from a therapeutic point of view, since it suggests that aptamers could be of use to rescue neutropenia in DC patients with TERC haploinsufficiency and short telomeres.

**Fig. 4: Aptamers rescue neutropenia in zebrafish models of DC and PN.**

In order to further support the usefulness of the clinical application of the terc-based aptamers, it was decided to test them in another zebrafish disease model of neutropenia. One of these models is a very rare autosomal recessive genodermatosis characterized by hypopigmentation or hyperpigmentation of different skin areas, skeletal defects and bone marrow alterations, called poikiloderma with neutropenia (PN)^31,32. PN is caused by mutations in the U6 biogenesis 1 (USB1) gene, a 3′‐5′ exonuclease able to post‐transcriptionally remove uridine and adenosine nucleosides from the 3′ end of the spliceosomal U6 small nuclear RNA. Nearly half of PN patients develop myelodysplastic syndrome and acute myeloid leukemia in the second decade of life, followed by bone marrow failure syndrome predisposing patients to cancer³³. The symptoms may be similar to those of DC, although the genetic causes are completely different³⁴. The treatment of this disease is based on alleviating the symptoms, so, in cases of neutropenia, GCSF is administered³⁵. To generate the zebrafish model of PN, genetic inactivation of usb1 with CRISPR-Cas9 was performed in the lyz:dsRED instead of the mpx:GFP zebrafish line, since it has been reported that usb1 deficiency results in alteration of several genes involved in neutrophil differentiation and development, including mpx³². It was confirmed that the genetic inhibition of usb1 results in a decreased number of neutrophils in zebrafish (Fig. 4b). Moreover, the administration of gcsf mRNA rescued neutropenia in this model (Fig. 4b), as occurs in PN patients. When the aptamers were used, interestingly, both T800 and T1000 were able to rescue neutropenia of the PN model, at similar levels to terc, while the negative controls CR7 and T1000_mut failed to do so (Fig. 4c). This result suggests that T800 and T1000 aptamers may be an effective treatment of human neutropenia that responds to GCSF treatment.

Aptamers rescue defective myeloid differentiation of mouse bone marrow cells and iPSCs from DC patients

T800 and T1000 zebrafish aptamers have been shown to increase the number of neutrophils in both healthy and neutropenic fish models, probably through the recruitment of RNA Pol II to myeloid gene promoters, such as spi1 and csf3b, thus enhancing their expression. These results, together with the absence of apparent side effects in the tested models, suggest their usefulness for treating human diseases. Consequently, the same aptamers, but based on the mouse or human telomerase RNA structure, were synthetized (Figs. S1c and S1d) and tested in mouse and human hematopoietic colony-forming cell assays, since the noncanonical function of TERC in myelopoiesis is highly conserved between mammals and zebrafish.

Unexpectedly, bone marrow cells from Terc^-/- showed weak, but statistically significant, myeloid differentiation defect compared with wild type and Tert^-/- cells (Fig. 5a and S3a). Addition of aptamer mT1000 increased the number of CFU-GM at the expenses of BFU-E in the 3 genotypes (Fig. 5a, S3a). We next evaluated the ability of aptamers to regulate myelopoiesis in humans using two iPSC lines derived from DC patients and one derived from a healthy donor (HD). iPSCs from DC patients harbored either a heterozygous mutation in the CR4/CR5 domain of TERC (nG319A)³⁶ or a heterozygous pathogenic missense point mutation in TERT (A716V)⁹. iPSCs were differentiated into hematopoietic cells through embryonic body (EB) formation, which are three-dimensional cell aggregates that can differentiate into cells of all three germ layers. Seven days after differentiation started, aptamers were added to the medium before carrying out colony-forming unit (CFU) assays and flow cytometry and gene expression analysis (Fig. 5b)³⁷. We firstly checked by RT-qPCR that aptamers were taken up by cells (Figs. S4a–S4c). As we recently reported⁹, iPSC harboring the TERC mutation showed impaired myelopoiesis (Fig. 5c, S3b). Strikingly, hT800 and hT1000 aptamers were both able to fully rescue the defective myeloid differentiation of TERC mutant iPSC by increasing the percentage of myeloid colonies (CFU-G/M/GM) to 50% (Fig. 5c). However, aptamer administration did not affect the percentage of hemogenic progenitors (HEPs) (CD31⁺/CD34⁺/CD45^-) (Fig. 5d), hematopoietic progenitors (HPC) (CD31^-/CD34⁺/CD45⁺) or mature blood cells (CD34^-/CD45⁺) (Figs. S3c, S3d, S3e). As the CFU potential robustly correlated with HEPs numbers³⁷ and aptamers did not change the number of progenitors, aptamers seem to act by favoring their differentiation into CFU-GM at the expenses of erythroid colonies (BFU-E). This is consistent with the ability of both hT800 and hT1000 aptamers to significantly rescue the reduced SPI1 transcript levels observed in the EB obtained from TERC-deficient iPSCs (Fig. 5e and S5a). However, although none of the aptamers significantly affected the transcript levels of GATA1, CSF3, TERT and TERC in the EBs, the administration of hT800 and hT1000 aptamers showed a tendency to increase the reduced mRNA levels found in the EB derived from TERC-deficient iPSCs (Figs. S5a–S5e). Together, these data further support the clinical application of the T800 domain-based aptamers to treat neutropenia of DC patients with mutations affecting the CR4/CR5 domain of TERC.

**Fig. 5: Aptamers rescue defective myelopoiesis in mouse and iPSC from DC patients.**

Discussion

Aptamers are of high interest for biomedicine application, such as treating cancer, immune diseases, acute myeloid leukemia, metabolic skeletal disorders or thrombosis, due to their ability to bind to specific biological molecules²⁴. Aptamers are selected through an in vitro process named Systematic Evolution of Ligands by Exponential enrichment (SELEX)³⁸. However, in our study, we took advantages of our extensive knowledge of TERC to design several aptamers. Hence, we have recently discovered that TERC behaves as a typical transcription factor, despite being a lncRNA⁹. On the one hand, it is able to recognize a specific binding sequence on the DNA and, on the other hand, to physically interact with RNA Pol II, the main component of the transcriptional machinery, in both zebrafish and humans. Reduced levels of TERC in human myeloid progenitors resulted in a decreased occupancy at transcription start sites of key myeloid genes by RNA Pol II, and thus, lower levels of myeloid transcripts⁹. Additionally, we have demonstrated that the CR4/CR5 domain is indispensable for this novel, noncanonical function of TERC, since a TERC molecule harboring a mutation in the CR4/CR5 domain found in DC patients is unable to regulate the expression of myeloid genes, whereas mutations of DC patients in other domains do not affect this function.

This crucial importance of the CR4/CR5 domain in myelopoiesis led us to try to develop new therapeutic strategies for DC patients, particularly those harboring mutations that affect the CR4/CR5 domain. For this purpose, an array of aptamers was designed based on the sequence of the CR4/CR5 domain. This tool was chosen because of its multiple advantages in terms of specificity, immunogenicity, cost and simplicity of synthesis²⁴. The T800 (CR4/CR5 full domain) and T1000 (P6.1 fork) were selected as therapeutic aptamers while the same sequence, but with the mutation present in DC patients (G305A for T1000 aptamer) was selected as control. As an additional control and to further demonstrate the relevance and autonomy of the CR4/CR5 domain to regulate myelopoiesis, we used the aptamer CR7 which consists on the ScaRNA domain sequence. Surprisingly, we observed that wild type CR4/CR5-based aptamers were able to increase the number of neutrophils and the expression of the master myeloid genes spi1b and csf3b in the zebrafish, just as terc did, while all control aptamers failed to do so. Importantly, the aptamers did not show toxic effects in zebrafish, and failed to induce inflammation and to alter neutrophil recruitment to wound. Mechanistically, aptamers function in the same way as full terc: (i) being able to increase the number of monocytes and neutrophils, without affecting the number of erythrocytes; (ii) increasing the promoter activity of csf3b in vivo by binding to tercbs; (iii) directly interacting with DNA in vitro and in vivo, such affinity being enhanced when tercbs were present; (iv) physically binding to RNA Pol II; and (v) they exerting all these effects independently of endogenous terc, since they did not affect the expression of terc and can perform their function in myelopoiesis in terc-deficient zebrafish models. These results not only show the therapeutical potential of these molecules to treat neutropenia but also demonstrate that the CR4/CR5 domain of terc regulates hematopoiesis independently of the rest of its domains.

To demonstrate the therapeutic use of the developed aptamers, bearing in mind that the noncanonical function of TERC in myelopoiesis is evolutionarily conserved⁹, homologous molecules were designed based on the mouse and human TERC sequences. It was found that both hT800 and hT1000 aptamers were able to slightly increase myelopoiesis in HD and TERT-deficient iPSCs but, more importantly, that they fully restored defective myelopoiesis in TERC-deficient iPSCs, probably by restoring normal transcript levels of SPI1, which were found to be lower, together with those of CSF3, in TERC-deficient EBs. These low levels of SPI1 and CSF3 in TERC-deficient EBs, but not in their TERT-deficient counterparts, is surprising because the culture medium contained GCSF, which apparently failed to restore defective granulopoiesis of TERC-deficient cells. Importantly, aptamer administration did not alter GATA1 mRNA levels or the number of HPCs in EBs, so it seems that TERC acts by fine-tuning SPI1 and GATA1 levels in HPCs in order to properly balance the production of blood cells, as do other master regulators, such as the inflammasome³⁹ and TIF1γ⁴⁰, among others. Importantly, these results, together with the observed lack of effect of aptamer administration in zebrafish erythropoiesis, suggest that aptamers might restore defective myelopoiesis in neutropenic patients without affecting erythropoiesis, an obvious concern in their clinical application. Although further studies in primary CD34⁺ cells from DC patients with mutation of the CR4/CR5 will surely further demonstrate its potential use to treat neutropenia, our study with zebrafish models of PN disease^31,32, strongly suggests its wide therapeutic potential in treating neutropenic congenital diseases caused by different mutations, beside those caused by mutations in TERC or telomeric proteins. Unexpectedly, although Terc-deficient bone marrow cells hardly recapitulated the myelopoiesis defects observed in zebrafish larvae and human iPSCs, the Terc-based aptamers were able to rescue these defects in all species.

An important observation of the present study is that although the T1000 aptamer showed reduced DNA-binding activity in vitro, both T800 and T1000 aptamers showed similar DNA-binding activity in vivo and ability to promote myelopoiesis in zebrafish and human. This suggests that the CR4/CR5 domain contains important nucleotides outside the P6.1 fork involved in the interaction with tercbs. While beyond the scope of the present study, we consider it would be interesting to map these nucleotides to further understand how TERC binds to its canonical target in the DNA¹⁰. It is also likely that other factors, which were only present in the in vivo assay, are important for the interaction between TERC and DNA. Whatever the outcome, the small size of T1000 makes it ideal as a potential therapeutic tool for clinical trial, since its tissue penetration capacity is greater, and the cost of its synthesis is much lower. In such clinical trials, it would also be interesting to compare the aptamers with the routine administration of GCSF, in terms of efficacy and cost, since Filgrastim (the generic name of commercial GCSF) and several biosimilar products, such as Lenograstim and Pegfilgrastim, have high cost (from €800 to €1800 range per patient and treatment cycle)⁴¹. Furthermore, we anticipate the usefulness of the developed aptamers for the treatment of chemotherapy-induced neutropenia in cancer patients, where post-chemotherapy GCSF treatment accounts for a large percentage (from 26 to 39%) of the total medical costs⁴².

In summary, based on our knowledge of the noncanonical function of TERC in the regulation of myelopoiesis, and in particular of its CR4/CR5 domain, the two aptamers that we have designed and characterized are able to perform the same function in myelopoiesis as the complete TERC molecule and to restore defective myelopoiesis in zebrafish, mouse and human models of congenital neutropenia caused by different genetic alterations, such as DC and PN.

Methods

Ethics statement

The performed experiments comply with the Guidelines of the European Union Council (Directive 2010/63/EU) and the Spanish RD 53/2013. Experiments and procedures were performed as approved by the Bioethical Committees of the University of Murcia (approval numbers #75/2014, #216/2014 and 395/2017).

Animals

Zebrafish (Danio rerio H.) specimens were obtained from the Zebrafish International Resource Center and mated, staged, raised and processed using standard procedures⁴³. The transgenic lines Tg(mpx::eGFP)ⁱ¹¹³ (mpx:GFP for simplicity)⁴⁴, Tg(mpeg1:eGFP)^gl22 (mpeg1:GFP for simplicity)⁴⁵, Tg(lcr:eGFP)^cz3325 (lcr:GFP for simplicity)⁴⁶ and Tg(lyz:dsRED)^nz50 (lyz:dsRED for simplicity)⁴⁷, were previously characterized.

Aptamers and RNA injection

Aptamers were synthesized as a single-stranded RNA oligos unmodified or with modifications to increase stability or to add biotin at 3’end by Sigma-Aldrich, or by Horizon Discovery in the case of T800 aptamer. The terc and csf3b RNAs were in vitro transcribed with the mMESSAGE mMACHINE kit (Ambion) and purified with phenol chloroform. Both mRNA and aptamers were mixed in a microinjection buffer (0.5 × Tango buffer and 0.05% phenol red solution) and microinjected into the yolk sac of one-cell stage embryos using a Narishige IM300 microinjector (0.5-1 nl per embryo). The amount of terc and csf3b injected was 200 and 100 pg/egg, respectively. The amount of aptamers injected was 605 pg/egg for T800, 337 pg/egg for CR7 and 138 pg/egg for T1000 and T1000_mut, in order to achieve the same number of molecules per egg in all the aptamers used.

Zebrafish blood cell count

Eggs of the one-cell stage from the transgenic lines mpx:GFP, mpeg1:GFP, lcr:GFP and lyz:dsRED were microinjected with the different aptamers or terc. Neutrophils (mpx:GFP or lyz:dsRed positive cells), and macrophages (mpeg1:eGFP positive cells) of the tail region of 48 or 72 hpf larvae were counted under a Leica M205 FA fluorescence stereo microscope. To quantify the erythrocytes (lcr:GFP positive cells), the larvae were photographed with Leica M205 FA fluorescence microscope equipped with a DFC365FX camera (Leica) and the total fluorescence intensity of the tail region was quantified with Leica Application Suite X (LAS X) software.

Zebrafish neutrophil staining

Zebrafish embryos (48 hpf) were fixed for 2 h at room temperature (RT) in 4% paraformaldehyde (PFA). Fixed larvae were briefly washed twice in PBS and incubated in 1:50 TSA Cyanine5 (TSA TM-Plus Fluorescein kit, Perkin Elmer, UK) without light for 10 min at 28 °C. Neutrophils were specifically fluorescein stained and then counted under a Leica M205 FA fluorescence stereo microscope equipped with a DFC365FX camera (Leica).

Aptamer quantification

Total RNA was extracted from larvae tails with TRIzol reagent (Thermo Fisher Scientific) using the Direct-zol RNA Miniprep kit (Zymo Research) and treated with DNAse following the manufacturer’s instructions. cDNA was generated with the miScript II RT kit (Qiagen), following the manufacturer’s instructions. Real-time qPCR was performed with a StepOnePlus instrument (Applied Biosystems), using miScriptSYBR Green PCR kit (Perfect Real Time) (Qiagen), containing a reverse common primer. Reaction mixtures were incubated for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C, and finally 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. For each sample, gene expression was normalized to U6 snRNA, using the comparative Ct method (2^-ΔΔCt). The forward primers used are shown in Table S1. In all cases, each PCR was performed with triplicate samples and repeated in every aptamer microinjection experiment.

Gene expression analysis

Total RNA was extracted from larvae tails with TRIzol reagent (Thermo Fisher Scientific) using Direct-zol RNA Miniprep kit (Zymo Research) following the manufacturer’s instructions. RNA was treated with DNase I, RNAsa free (Qiagen) on the column. A SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen) was used to synthesize first-strand cDNA following the manufacturer’s instructions. Real-time PCR was performed with a StepOnePlus instrument (Applied Biosystems) using SYBR® Premix Ex Taq™ (Perfect Real Time) (Takara). Reaction mixtures were incubated for 30 s (sec) at 95 °C, followed by 40 cycles of 5 sec at 95 °C, 20 sec at 60 °C, and finally a melting curve protocol. Ribosomal protein S11 (rps11) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) content in each sample was used for normalization of zebrafish or human mRNA expression, respectively, using the comparative Ct method (2-∆Ct). The primers used are shown in Table S1. In all cases, each PCR was performed with triplicate samples and repeated with at least two independent samples.

Recruitment assay

At 3 dpf, Tg(mpx::GFP) injected with T1000 or T1000mut aptamers and uninjected as control larvae were anesthetized in tricaine and complete transection of the tail was performed with a disposable sterile scalpel. Then, they were mounted in 1% (wt/vol) low-melting-point agarose dissolved in egg water. The images were captured at the selected times while animals were kept in the agarose matrixes with the added medium at 28.5 °C. All images were acquired with the integrated camera on the stereomicroscope and were used for subsequently counting the number of neutrophils recruited to the wound area, established between the arterio-venous loop and the end of the tail.

Telomerase activity assay

A real-time quantitative telomeric repeat amplification protocol (Q-TRAP) analysis was performed to measure telomerase activity⁴⁸. Briefly, proteins were extracted from whole 48 hpf embryos, previously microinjected with RNA, aptamers or 20 µM BIBR1532 (telomerase inhibitor, Santa Cruz Biotechnology, #203843), using ice-cold TRAPeze 1X CHAPS Lysis Buffer (Sigma Aldrich). A real-time Q-TRAP was performed with 0.1 μg protein extracts. To make the standard curve, a 1:10 dilution series of positive telomerase sample (HeLa cells) was used. Control samples were obtained by treating the cell extracts with 1 μg RNase at 37 °C for 20 min. Data was collected and converted into Relative Telomerase Activity (RTA) units by calculating: RTA of sample=10^{(Ct sample−γint)/slope}. The standard curve obtained was y = −3.2295x + 23.802.

In vivo luciferase assay

pCMV-Renilla plasmid, containing Renilla luciferase (Promega) and pGL3basic vector constructs that contained the csf3b promoter region of zebrafish followed by firefly luciferase, were microinjected together with indicated aptamers or terc into the yolk sac of single-cell stage embryos. After 48 h, tail sections were obtained, pooled (20 tails per pool), homogenized with a pellet pestle and centrifuged for 3 min at 10,000 g to remove cellular debris. Then, extracts were assayed for firefly and Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega) on a Luminometer Optocomp I (MGM Instruments). The results were normalized with the Renilla activity⁴⁹.

In vitro DNA binding assay

One hundred bp sense and antisense 3´ biotin-tagged-DNA probes of zebrafish csf3b promoter region encompassing terc-binding sites (Sigma-Aldrich) were designed to measure the capacity of terc or aptamers to bind to these sequences⁹. For annealing, 25 µM of each probe were incubated in annealing buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.2 mM EDTA) at 95 °C for 4 min, 10 min at 70 °C and they were slowly cooled down to RT for 20 min to allow annealing. Then, dsDNA probes were bound to 10 µl of Dynabeads MyOne Streptavidin C1 magnetic beads (Invitrogen) for 15 min at RT. Probe excess was removed by washing beads 2 times with annealing buffer for 5 min. Then, 50 ng of luciferase (Promega), terc and aptamers were added and incubated at RT for 30 min in rotation. The beads-dsDNA-RNA complexes were washed 3 times at RT for 10 min and RNA was eluted by incubation in water at 95 °C for 5 min. The eluted RNAs were reverse transcribed with SuperScript IV VILO Master Mix (Invitrogen) or miScript II RT kit (Qiagen) following the manufacturer’s instructions, for terc and luciferase or for aptamers, respectively. The samples were subjected to quantitative-PCR (qPCR) for luciferase, terc and aptamers detection, under the same conditions. To be able to compare the expression cycles of terc, aptamers and luciferase, normalization was carried out previously. Normalization consisted of a primers efficiency calibration curve starting from 1 ng/µl of RNA, and serial dilutions of 0.1 ng/µl, 0.01 ng/µl and 0.001 ng/µl of every RNA. Luciferase primers are more efficient than terc primers in 1.3 cycles, CR7 aptamer in 3.8 cycles, T800 aptamer in 19.2 cycles and T1000 and T1000_mut aptamers in 18.7 cycles.

Aptamer-based chromatin immunoprecipitation

A Chromatin Isolation by RNA Purification (ChIRP) protocol⁹ was performed with some modifications. Briefly, 5×106 HL60 cells were treated with 3´biotinylated-human aptamers (1 uM final concentration) in the culture medium for 48 h. Cells were washed in PBS, crosslinked with 1% gultaraldehyde for 10 min at room temperature, followed by incubation with 1/10 volume of 1,25 M glycine and lysed in ChIRP lysis buffer. Chromatin was then sonicated to a DNA fragment size of 100-500 bp in a Bioruptor Pico (Diagenode) and 100 ug of chromatin was incubated for 1 h with with Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Thermo Fisher Scientific) at 37 °C. Beads:biotin-aptamer:chromatin adducts were captured by magnets (Thermo Fisher Scientific) and washed five times with ChIRP washing buffer. Beads were resuspended in DNA elution buffer, and the DNA was eluted with a cocktail of RNase A (Sigma-Aldrich) and RNase H (Epicenter). Eluted chromatin was treated with proteinase K for 45 min at 65 °C. DNA was then extracted with equal volume of phenol:chloroform:isoamyl alcohol and precipitated with ethanol overnight at −80 °C. Eluted DNA was subjected to qPCR for detection of tercbs-containing CSF2 promoter or 3'UTR SPI1 gene chromatin fragments.

RNA pulldown

RNA pulldown experiments were performed as described⁵⁰, with some modifications⁹. Biotin-labeled RNAs were in vitro transcribed using Biotin 3´ End DNA Labeling Kit (Thermo Scientific), to incorporate 1-3 biotinylated ribonucleotides onto 3´ end of DNA/RNA strands, following the manufacturer’s instructions. Three micrograms of biotinylated RNA were heated to 70 °C for 5 min and put on ice for 2 min. An equal volume of 2x RNA structure buffer (20 mM Tris pH 7, 0.2 M KCl, 20 mM MgCl₂) was added and then shifted to RT to allow RNA secondary structure formation. Folded RNAs were incubated for 1 h at 4 °C with rotation with 60 µl of washed Dynabeads MyOne Streptavidin C1 magnetic beads (Invitrogen). Protein extract from 5 dpf larvae was obtained by homogenization in RIP buffer (25 mM Tris pH 7.4, 150 mM KCl, 0.5 mM DTT, 0.5% NP-40), centrifuged for 20 min at 4 °C and the supernatant was pre-cleared 1 h with 30 µl of beads at 4 °C under rotation. Then, three miligrams of pre-cleared protein were incubated with biotinylated RNAs-beads complexes for 4 h at 4 °C with rotation. Complexes were magnet-captured, washed with RIP buffer five times at 4 °C for 5 min and boiled in 2x Laemmli buffer (Sigma) at 90 °C for 10 min for protein elution. Eluted proteins were subjected to polyacrylamide gel electrophoresis, wet transferred to a nylon membrane (GE Healthcare) and analyzed by Western blot. Membranes were incubated for 1 h with TTBS (Tween-Tris-buffered saline) containing 5% (w/v) skimmed dried milk powder and immunoblotted using anti-phospho-Serine 5 RNA polymerase II CTD repeat YSPTSPS mouse antibodies (dilution 1:1000) 16 h at 4 °C (pS5 RNA pol II, ab5408, Abcam). Blots were then washed with TTBS and incubated for 1 h at room temperature with the secondary HRP-conjugated antibody (dilution 1:1000) in 5% (w/v) skimmed milk in TTBS. After repeated washes, the signal was detected with the enhanced chemiluminescence reagent and ChemiDoc XRS (Biorad).

Zebrafish model of poikiloderma with neutropenia

To generate a zebrafish model of poikiloderma with neutropenia, the usb1 (U6 SnRNA Biogenesis Phosphodiesterase 1) gene^31,32 was inhibited by CRISPR-Cas9 technology. CRISPR RNA (crRNA) was obtained from IDT with the following target sequence was used: usb1 5′-GGAAGCTCTTCATCACCTTCAGG-3′. It was resuspended in duplex buffer at 100 μM and 1 μl was incubated with 1 μl of trans-activating CRISPR RNA (tracrRNA, 100 μΜ) at 95 °C for 5 min and 5 min at RT to form the complex. One μl of these complexes was mixed with 0.25 μl of recombinant Cas9 (10 mg/ml) and 3.75 μl of duplex buffer. The crRNA mix was then microinjected into the yolk sac of one-cell stage zebrafish embryos using a microinjector (Narishige) (1 nl per embryo). Neutrophils were counted at 3 dpf.

iPSC culture and differentiation towards hematopoietic lineage and CFU assay

iPSC were obtained by Dr Sunnet Agarwal (Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.) who has custody of confirmation of IRB approval and patient consents.

iPSC lines were maintained undifferentiated in a 6 cm plates treated with Matrigel (Corning) and mTeSR™ Plus medium (Stem Cell Technologies). The medium was changed daily or every two days, and cells were split weekly by dissociation with 200 U/ml of collagenase IV (Invitrogen). iPSC cultures were visualized daily by phase-contrast microscopy.

For hematopoietic differentiation, undifferentiated iPSC at 70-80% confluence were treated with Matrigel (Corning) 24 h before starting differentiation. To generate EBs, the iPSC were treated with collagenase IV and scraped off. They were then transferred to 6-well low-attachment plates (Corning) to allow EB formation by incubation in differentiation medium consisting of KnockOut™ Dulbecco’s modified Eagle’s medium (ThermoFisher) supplemented with 20% non-heat-inactivated fetal bovine serum, 1% nonessential amino acids, 1 mM glutamine and 0.1 mM β-mercaptoethanol. The medium was changed the following day (day 1) with the same differentiation medium supplemented with hematopoietic cytokines: 300 ng/ml stem cell factor (R&D), 300 ng/ml FMS-like tyrosine kinase-3 ligand (R&D), 10 ng/ml interleukin-3 (IL-3, R&D), 10 ng/ml IL-6 (R&D), 50 ng/ml GM-CSF (R&D) and 25 ng/ml bone morphogenetic protein-4 (Miltenyi)^51,52,53. At day 7, aptamers are added to the medium and refreshed every 2 days. At day 15 of development, EBs were dissociated, and single-cell suspensions were stained with anti-CD34-fluorescein isothiocyanate, anti-CD31-phycoerythrin and anti-CD45-allophycocyanin antibodies (all from Becton Dickinson) and analyzed by flow cytometry.

CFU assays were performed by plating 100,000 cells from EBs at day 15 into methylcellulose H4434 culture medium (Stem Cell Technologies). The cells were incubated at 37 °C in a 5% CO₂ humidified atmosphere and colonies were counted at day 14 of the CFU assay using standard morphological criteria^54,55,56. The iPSC workflow is depicted in Fig. 5b.

Modeling of three-dimensional structures of aptamers and TERC

Prediction and modelling of tertiary RNA structure using the RNAComposer system (http://rnacomposer.cs.put.poznan.pl/)^57,58,59,60. The structure was processed with Jmol application, an open-source Java viewer for chemical structures in 3D.

Statistical analysis

Statistical analysis was performed by using GraphPad Prism 8. Data were analyzed by analysis of variance (ANOVA) or mixed-effect analysis (see Figure legends for further details).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data are provided with this paper. All data supporting the findings described in this manuscript are available in the article and in the Supplementary Information and from the corresponding author upon request. Source data are provided with this paper.

References

Blackburn, E. H. Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 579, 859–862 (2005).
Article CAS PubMed Google Scholar
Wyatt, H. D., West, S. C. & Beattie, T. L. InTERTpreting telomerase structure and function. Nucleic Acids Res. 38, 5609–5622 (2010).
Article CAS PubMed PubMed Central Google Scholar
Zhang, Q., Kim, N. K. & Feigon, J. Architecture of human telomerase RNA. Proc. Nat. Acad. Sci. USA 108, 20325–20332 (2011).
Article CAS PubMed PubMed Central ADS Google Scholar
Nguyen, T. H. D. et al. Cryo-EM structure of substrate-bound human telomerase holoenzyme. Nature 557, 190–195 (2018).
Article CAS PubMed PubMed Central ADS Google Scholar
Finkel, T., Serrano, M. & Blasco, M. A. The common biology of cancer and ageing. Nature 448, 767–774 (2007).
Article CAS PubMed ADS Google Scholar
Cong, Y. & Shay, J. W. Actions of human telomerase beyond telomeres. Cell Res. 18, 725–732 (2008).
Article CAS PubMed Google Scholar
Martínez, P. & Blasco, M. A. Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins. Nat. Rev. Cancer 11, 161–176 (2011).
Article PubMed Google Scholar
Alcaraz-Perez, F. et al. A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish. Nat. Commun. 5, 3228 (2014).
Article PubMed ADS Google Scholar
García-Castillo, J. et al. Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis. Proc. Nat. Acad. Sci. 118, e2015528118 (2021).
Article PubMed PubMed Central Google Scholar
Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. & Chang, H. Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol Cell 44, 667–678 (2011).
Article CAS PubMed PubMed Central Google Scholar
Dale, D. C. & Bolyard, A. A. An update on the diagnosis and treatment of chronic idiopathic neutropenia. Curr. Opin. Hemat. 24, 46–53 (2017).
Article CAS Google Scholar
Spoor, J., Farajifard, H. & Rezaei, N. Congenital neutropenia and primary immunodeficiency diseases. Crit. Rev. Oncology/hematology 133, 149–162 (2019).
Article Google Scholar
Lalami, Y. & Klastersky, J. Impact of chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) on cancer treatment outcomes: An overview about well-established and recently emerging clinical data. Crit. Rev. Oncol./hematol. 120, 163–179 (2017).
Article PubMed Google Scholar
Vulliamy, T. J. & Dokal, I. Dyskeratosis congenita: the diverse clinical presentation of mutations in the telomerase complex. Biochimie 90, 122–130 (2008).
Article CAS PubMed Google Scholar
Dokal, I. Dyskeratosis congenita. Hematol. Am. Soc. Hematol. Educ. Program 2011, 480–486 (2011).
Article Google Scholar
Mason, P. J. & Bessler, M. The genetics of dyskeratosis congenita. Cancer Genet. 204, 635–645 (2011).
Article CAS PubMed PubMed Central Google Scholar
Vulliamy, T. J. et al. Differences in disease severity but similar telomere lengths in genetic subgroups of patients with telomerase and shelterin mutations. PLoS One 6, e24383 (2011).
Article CAS PubMed PubMed Central ADS Google Scholar
Dale, D. C. How I diagnose and treat neutropenia. Curr. Opin. Hematol. 23, 1–4 (2016).
Article PubMed PubMed Central Google Scholar
Mehta, H. M., Malandra, M. & Corey, S. J. G-CSF and GM-CSF in Neutropenia. J. Immunol. 195, 1341–1349 (2015).
Article CAS PubMed Google Scholar
Yilmaz, K., Inaloz, H. S., Unal, B. & Guler, E. Dyskeratosis congenita with isolated neutropenia and granulocyte colony-stimulating factor treatment. Int. J. Dermatol. 41, 170–172 (2002).
Article PubMed Google Scholar
Erduran, E., Hacisalihoglu, S. & Ozoran, Y. Treatment of dyskeratosis congenita with granulocyte-macrophage colony-stimulating factor and erythropoietin. J. Ped. Hematol./Oncol. 25, 333–335 (2003).
Article Google Scholar
Alter, B. P., Gardner, F. H. & Hall, R. E. Treatment of dyskeratosis congenita with granulocyte colony-stimulating factor and erythropoietin. Br. J. Haematol. 97, 309–311 (1997).
Article CAS PubMed Google Scholar
Oehler, L. et al. Effective stimulation of neutropoiesis with rh G-CSF in dyskeratosis congenita: a case report. Ann. Hematol. 69, 325–327 (1994).
Article CAS PubMed Google Scholar
Zhuo, Z. et al. Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine. Int. J. Mol. Sci. 18, 2142 (2017).
Article Google Scholar
Ng, E. W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123–132 (2006).
Article CAS PubMed Google Scholar
Zhou, J. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat. Rev. Drug Discov. 16, 181–202 (2017).
Article CAS PubMed Google Scholar
Carroll, K. A. & Ly, H. Telomere dysfunction in human diseases: the long and short of it! Int. J. Clin. Exp. Pathol. 2, 528–543 (2009).
CAS PubMed PubMed Central Google Scholar
Ni, S. et al. Chemical modifications of nucleic acid aptamers for therapeutic purposes. Int. J. Mol. Sci. 18, 1683 (2017).
Article PubMed PubMed Central Google Scholar
Zhu, G. & Chen, X. Aptamer-based targeted therapy. Adv. Drug Del. Rev. 134, 65–78 (2018).
Article CAS Google Scholar
Kovacevic, K. D., Gilbert, J. C. & Jilma, B. Pharmacokinetics, pharmacodynamics and safety of aptamers. Adv. Drug Del. Rev. 134, 36–50 (2018).
Article CAS Google Scholar
Colombo, E. A. et al. A zebrafish model of Poikiloderma with Neutropenia recapitulates the human syndrome hallmarks and traces back neutropenia to the myeloid progenitor. Sci. Rep. 5, 15814 (2015).
Article CAS PubMed PubMed Central ADS Google Scholar
Patil, P., Uechi, T. & Kenmochi, N. Incomplete splicing of neutrophil-specific genes affects neutrophil development in a zebrafish model of poikiloderma with neutropenia. RNA Biol. 12, 426–434 (2015).
Article PubMed PubMed Central Google Scholar
Negri, G. et al. Expanding the role of the splicing USB1 gene from Poikiloderma with Neutropenia to acquired myeloid neoplasms. Br. J. Haematol. 171, 557–565 (2015).
Article CAS PubMed Google Scholar
Walne, A. J. et al. Marked overlap of four genetic syndromes with dyskeratosis congenita confounds clinical diagnosis. Haematologica 101, 1180–1189 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wang, L., Clericuzio, C. & Larizza, L. Poikiloderma with Neutropenia. In GeneReviews® (eds. Adam, M.P. et al.) (Seattle (WA), 2017).
Boyraz, B., Bellomo, C. M., Fleming, M. D., Cutler, C. S. & Agarwal, S. A novel TERC CR4/CR5 domain mutation causes telomere disease via decreased TERT binding. Blood 128, 2089–2092 (2016).
Article CAS PubMed PubMed Central Google Scholar
Ramos-Mejia, V. et al. Nodal/Activin signaling predicts human pluripotent stem cell lines prone to differentiate toward the hematopoietic lineage. Mol. Ther. 18, 2173–2181 (2010).
Article CAS PubMed PubMed Central Google Scholar
Wu, Y. X. & Kwon, Y. J. Aptamers: The “evolution” of SELEX. Methods 106, 21–28 (2016).
Article CAS PubMed Google Scholar
Tyrkalska, S. D. et al. Inflammasome regulates hematopoiesis through cleavage of the master erythroid transcription factor GATA1. Immunity 51, 50–63.e55 (2019).
Article CAS PubMed PubMed Central Google Scholar
Monteiro, R., Pouget, C. & Patient, R. The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1gamma. EMBO J. 30, 1093–1103 (2011).
Article CAS PubMed PubMed Central Google Scholar
Hadji, P., Kostev, K., Schroder-Bernhardi, D. & Ziller, V. Cost comparison of outpatient treatment with granulocyte colony-stimulating factors (G-CSF) in Germany. Int. J. Clin. Pharmacol. Therap. 50, 281–289 (2012).
Article Google Scholar
Tilleul, P., Jacot, W., Emery, C., Lafuma, A. & Gourmelen, J. Management and cost analysis of cancer patients treated with G-CSF: a cohort study based on the French national healthcare insurance database. J. Med. Econ. 20, 1261–1267 (2017).
Article PubMed Google Scholar
Widrick, J. J. et al. An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish. PloS one 13, e0199712 (2018).
Article PubMed PubMed Central Google Scholar
Renshaw, S. A. et al. A transgenic zebrafish model of neutrophilic inflammation. Blood 108, 3976–3978 (2006).
Article CAS PubMed Google Scholar
Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. & Lieschke, G. J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49–e56 (2011).
Article CAS PubMed PubMed Central Google Scholar
Ganis, J. J. et al. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Dev. Biol. 366, 185–194 (2012).
Article CAS PubMed PubMed Central Google Scholar
Hall, C., Flores, M. V., Storm, T., Crosier, K. & Crosier, P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42 (2007).
Article PubMed PubMed Central Google Scholar
Herbert, B. S., Hochreiter, A. E., Wright, W. E. & Shay, J. W. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nat. Protocols 1, 1583–1590 (2006).
Article CAS PubMed Google Scholar
Alcaraz-Perez, F., Mulero, V. & Cayuela, M. L. Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos. BMC Biotechnol. 8, 81 (2008).
Article PubMed PubMed Central Google Scholar
Tsai, M. C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010).
Article CAS PubMed PubMed Central ADS Google Scholar
Wang, L. et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 21, 31–41 (2004).
Article CAS PubMed Google Scholar
Menendez, P., Wang, L., Chadwick, K., Li, L. & Bhatia, M. Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors. Mol. Ther. 10, 1109–1120 (2004).
Article CAS PubMed Google Scholar
Wang, L. et al. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J. Exp. Med. 201, 1603–1614 (2005).
Article CAS PubMed PubMed Central Google Scholar
Bueno, C. et al. Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells. Carcinogenesis 30, 1628–1637 (2009).
Article CAS PubMed Google Scholar
Menendez, P., del Canizo, M. C. & Orfao, A. Immunophenotypic characteristics of PB-mobilised CD34+ hematopoietic progenitor cells. J. Biol. Regul. Homeost. Agents 15, 53–61 (2001).
CAS PubMed Google Scholar
Catalina, P. et al. Genetic stability of human embryonic stem cells: A first-step toward the development of potential hESC-based systems for modeling childhood leukemia. Leuk. Res. 33, 980–990 (2009).
Article CAS PubMed Google Scholar
Antczak, M. et al. New functionality of RNAComposer: an application to shape the axis of miR160 precursor structure. Acta Biochimica Polonica 63, 737–744 (2016).
CAS PubMed Google Scholar
Biesiada, M., Purzycka, K. J., Szachniuk, M., Blazewicz, J. & Adamiak, R. W. Automated RNA 3D structure prediction with RNAComposer. Meth. Mol. Biol. 1490, 199–215 (2016).
Article CAS Google Scholar
Popenda, M. et al. Automated 3D structure composition for large RNAs. Nucleic Acids Res. 40, e112 (2012).
Article CAS PubMed PubMed Central Google Scholar
Rybarczyk, A. et al. New in silico approach to assessing RNA secondary structures with non-canonical base pairs. BMC Bioinform. 16, 276 (2015).
Article Google Scholar

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Acknowledgements

We warmly acknowledge Cynthia Cabello, Rosa Serrano, Maria Inmaculada Fuentes and Pedro. J. Martínez for their excellent technical assistance. We also thank Profs. S.A. Renshaw, P. Crosier and LI Zon for the fish lines Tg(mpx::eGFP), Tg(lyz:dsred) and Tg(lcr:GFP), respectively, and Prof. S Agarwal for the iPSC lines. This work was supported by the Spanish Ministry of Science and Innovation (grants iscIII PI19/00188 and PI22/00861 to MLC and 2020-113660RB-I00 to VM, and Juan de la Cierva-Formación postdoctoral contract to FAP), all co-funded with Fondos Europeos de Desarrollo Regional), Fundación Séneca-Murcia (grants 20793/PI/18 and 21887/PI/22 to VM), Fundación Ramón Areces (grant to MLC), the University of Murcia (postdoctoral PIT2 contract to DGM and predoctoral contract to EMB and MFL), Spanich Ministry of Universities (predoctoral contract to ENS) and Consejería de Sanidad de la Región de Murcia (postdoctoral contract to FAP and JGC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

Grupo de Telomerasa, Cáncer y Envejecimiento, Hospital Clínico Universitario Virgen de la Arrixaca, 30120, Murcia, Spain
Elena Martínez-Balsalobre, Jesús García-Castillo, Diana García-Moreno, Elena Naranjo-Sánchez, Miriam Fernández-Lajarín, Francisca Alcaraz-Pérez & María L. Cayuela
Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, 30120, Murcia, Spain
Elena Martínez-Balsalobre, Jesús García-Castillo, Diana García-Moreno, Elena Naranjo-Sánchez, Miriam Fernández-Lajarín, Francisca Alcaraz-Pérez, Victoriano Mulero & María L. Cayuela
Departamento de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, 30100, Murcia, Spain
Elena Martínez-Balsalobre, Diana García-Moreno, Elena Naranjo-Sánchez, Miriam Fernández-Lajarín, Francisca Alcaraz-Pérez & Victoriano Mulero
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, 28029, Madrid, Spain
Elena Martínez-Balsalobre, Diana García-Moreno, Elena Naranjo-Sánchez, Miriam Fernández-Lajarín, Francisca Alcaraz-Pérez, Victoriano Mulero & María L. Cayuela
Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
María A. Blasco

Authors

Elena Martínez-Balsalobre
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Jesús García-Castillo
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Elena Naranjo-Sánchez
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Francisca Alcaraz-Pérez
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Victoriano Mulero
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Contributions

M.L.C. conceived the study; J.G.C., D.G.M., F.A.P., V.M. and M.L.C. designed the research; M.A.B. provided mouse models. E.M.B., D.G.M., J.G.C.,E.N.S., M.F.L. and F.A.P. performed the research; E.M.B., D.G.M., J.G.C., F.A.P., E.N.S., M.F.L., V.M. and M.L.C. analyzed data; and E.M.B., F.A.P., V.M. and M.L.C. wrote the manuscript with minor contribution from other authors.

Corresponding authors

Correspondence to Francisca Alcaraz-Pérez, Victoriano Mulero or María L. Cayuela.

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Competing interests

A patent for the use of TERC-based aptamers to treat blood diseases has been registered by the Universidad de Murcia, Instituto Murciano de Investigación Biosanitaria and CIBER-ER (EP20383104). E.M.B., D. G.M., J.G.C., F.A.P., V.M., and M.L.C. are involved in the patent. The other authors declare no competing interests.

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Martínez-Balsalobre, E., García-Castillo, J., García-Moreno, D. et al. Telomerase RNA-based aptamers restore defective myelopoiesis in congenital neutropenic syndromes. Nat Commun 14, 5912 (2023). https://doi.org/10.1038/s41467-023-41472-7

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Received: 04 October 2021
Accepted: 06 September 2023
Published: 22 September 2023
DOI: https://doi.org/10.1038/s41467-023-41472-7

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Subjects

Abstract

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Branched chemically modified poly(A) tails enhance the translation capacity of mRNA

Introduction

Results

Aptamers were designed based on CR4/CR5 domain of TERC

Aptamers increase myelopoiesis, without affecting erythropoiesis, in zebrafish

Aptamers physically bind to DNA and to RNA pol II

Aptamers rescue neutropenia in zebrafish disease models of dyskeratosis congenita and poikiloderma with neutropenia

Aptamers rescue defective myeloid differentiation of mouse bone marrow cells and iPSCs from DC patients

Discussion

Methods

Ethics statement

Animals

Aptamers and RNA injection

Zebrafish blood cell count

Zebrafish neutrophil staining

Aptamer quantification

Gene expression analysis

Recruitment assay

Telomerase activity assay

In vivo luciferase assay

In vitro DNA binding assay

Aptamer-based chromatin immunoprecipitation

RNA pulldown

Zebrafish model of poikiloderma with neutropenia

iPSC culture and differentiation towards hematopoietic lineage and CFU assay

Modeling of three-dimensional structures of aptamers and TERC

Statistical analysis

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

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Competing interests

Peer review

Peer review information

Additional information

Supplementary Information

Supplementary Information

Reporting Summary

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Source Data

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