Patent Application
20250066784
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing xml file entitled: 128-1280-CIP_SeqListing.xml; size: 36,523 bytes; and date of creation: Sep. 18, 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52 (e) (5).
The present invention relates to an oligonucleotide comprising one of the following sequence: (TTAGGG) SEQ ID No. 1. (TAGGGT) SEQ ID No. 2, (AGGGTT) SEQ ID No. 3, (GGGTTA) SEQ ID No. 4, (GGTTAG) SEQ ID No. 5 or (GTTAGG) SEQ ID No. 6 or a complementary sequence thereof or a fragment or a variant or a mixture thereof for use in the treatment and/or prevention of a disease characterized by alternative lengthening of telomeres or a non-cancer condition associated with telomere dysfunction and relative pharmaceutical compositions and to relative pharmaceutical composition and method.
DNA is a unique type of molecule in a cell in that, if damaged, it cannot be replaced. The so-called “DNA damage response” (DDR) is a coordinated set of evolutionarily-conserved events that, when triggered upon DNA damage detection, arrests the cell cycle (DNA damage checkpoint function) and coordinates DNA repair (Jackson and Bartek, 2009). DNA damage is a physiological event. Ageing and cancer are probably the two best examples in mammals that highlight the relevance of DNA damage accumulation, DDR activation and its consequences. Significant contribution has been made to the understanding of DDR engagement both in ageing and in cancer (d'Adda di Fagagna, 2008; Jackson and Bartek, 2009).
More recently, the present inventors have unveiled and reported that full DDR activation depends on RNA molecules. They observed that DNA double-strand breaks (DSBs) trigger the local generation of non-coding RNAs at the site of DNA damage carrying the sequence surrounding the damaged site. They have also shown that these RNA (the authors called them DDRNAs) are essential for DDR activation. Indeed, removal of DDRNA by RNase A treatment inhibits DDR activation, and DDR can be fully restored by the addition of chemically-synthesized DDRNA carrying the sequence surrounding the damaged site but not other sequences (Francia et al., 2012).
Several studies have shown that RNA functions can be inhibited by the use of inhibitory antisense oligonucleotides (ASOs) that act by pairing with target RNAs and thus impairing their functions. Their use is successfully reaching the clinical stage (Janssen et al., 2013; Li and Rana, 2014; Monteleone et al., 2015; Stenvang et al., 2012). Cancer cells must preserve telomere length and counteract natural telomere attrition to retain unlimited proliferative potential. Most cancers cells achieve this by reactivating telomerase expression, an enzyme that elongates short telomeres. However, 10-15% of all human tumors, with a higher incidence in, but not exclusively, osteosarcomas, soft tissue sarcomas, primary brain tumors, glioblastoma multiforme (GBM) and neuroblastomas maintain telomere length by the so-called Alternative Lengthening of Telomeres (ALT) mechanism based on homologous recombination (HR) among telomeres (Cesare and Reddel, 2010; Durant, 2012; Henson and Reddel, 2010).
Although ALT tumors may carry different genetic mutations causative of their transformed and malignant state, most ALT cancer cells show mutations in DAXX and ATRX genes, thus making possible the classification of tumors as ALT by common genetic analyses (Heaphy et al., 2011).
ALT mechanisms have never been reported in healthy/normal cells (Cesare and Reddel, 2010). However, in addition to cancer, ALT mechanisms have been reported in EBV-infected cells (Kamranvar et al., 2013) highlighting the possibility that other viral infections may trigger such mechanisms.
The use of telomerase inhibitors in cancer therapy has been investigated widely (Ruden and Puri, 2013); however, an intrinsic limitation of this approach is that lack of telomerase activity in tumors can lead to the selection of ALT positive clones, as reported in (Hu et al., 2012). Differently, so far, no reversal from ALT to telomerase mechanisms of telomere maintenance have been reported.
ALT cells show chronic DDR activation at telomeres indicating the dysfunctional nature of telomeres in such cells. Critically short/dysfunctional telomeres are then elongated/“repaired” by DDR mechanism engaging HR pathways. ALT-associated PML bodies (APBs) contain telomeric DNA and DDR factors and are a known biomarker of ALT cells (Cesare and Reddel, 2010; Yeager et al., 1999). HR is a DNA repair mechanism part of DDR and it has been shown to be necessary for the maintenance of telomeres in ALT cells. The MRN (MRE11/RAD50/NBS1) complex is a key DDR factor involved in the HR pathway. Indeed, its inactivation by either overexpression of the inhibitory protein SP100 or short hairpin-mediated knockdown, determines the inhibition of the telomere maintenance specifically in the ALT-positive cells (Jiang et al., 2005; Jiang et al., 2007; Zhong et al., 2007). RNA interference-mediated depletion of the SMC5/6 complex, which promotes HR-mediated repair of DSBs, results in shortened telomeres and cellular senescence in ALT cells (Potts and Yu, 2007). RPA binds to single strand DNA during the initial phase of HR and its downregulation through RNA interference causes impairment in ALT activity (Jiang et al., 2007). More recently, an ATR small molecule inhibitor has been shown to prevent cell growth specifically of ALT-positive cells (Flynn et al., 2015), although this specificity seems to be broader, including non-ALT cancer cells and only in combination with CHK1 inhibitors (Sanjiv et al., 2016). Consistently, another recent report suggested that the cell-type specificity of the ATR inhibitors is more related to the cell confluency than to the mechanism of telomere maintenance (http://biorxiv.org/content/early/2016/06/04/053280).
The inhibition of DDR and consequent recombination events at telomeres in ALT cells could be exploited to conceive novel potential therapeutic avenues for anticancer treatments. Consistently, as mentioned above, ALT cells have been proposed to be highly sensitive to the inhibition of the ATR kinase (Flynn et al., 2015), a protein involved in DDR signaling in the context of DNA repair by HR, although its specificity has been recently put into question (Sanjiv et al., 2016).
Telomere Biology Disorders (TBDs) or telomeropathies comprise a wide range of disorders that develop as a consequence of defects in telomere maintenance homeostasis that leads to telomere shortening and dysfunction. TBDs can be considered accelerated premature aging syndromes, since telomeres also become shortened and dysfunctional with aging, leading to a plethora of manifestations common to both (Roka, 2023; Rossiello, 2023).
Conditions characterized by telomeric DNA damage often leading to a progeric phenotype (premature ageing), are expected to be associated with the generation of DDRNAs with a telomeric sequence. Other examples of age-related diseases include Idiopathic pulmonary fibrosis (IPF), Alzheimer's disease (AD), and several hematopoietic and immune manifestations such as dyskeratosis congenita, aplastic anemia, altered myeloid progenitor differentiation, and bone marrow failure.
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive fibrosis and loss of alveolar epithelial cell integrity. It affects approximately 3 million people worldwide and, being an age-associated disease, its incidence is expected to increase in the near future with the aging population (Maher et al, 2021).
IPF patients have generally a poor prognosis (4 years median survival post diagnosis) and the only presently available therapy consists in the use of the anti-fibrotic drugs nintedanib and pirfenidone, which however can only slow down the disease progression, with many side effects (Hughes et al, 2016). Cure so far can only be provided by lung transplantation, with the known risks and costs. Therefore, there is a compelling need for a safer and more efficacious approach to treat this disease.
Despite its name, suggesting an unknown cause, an increasing body of evidence indicates that IPF is associated with and caused by telomere dysfunction, reviewed in (Armanios, 2012).
Indeed, 37% of familial cases and 25% of sporadic cases show leukocyte telomere lengths below the tenth percentile for their age (Cronkhite et al. Am. J. Respir. Crit. Care Med., 2008) and patients often show germline mutations in genes involved in telomere maintenance, including telomerase Armanios, et al. N. Engl. J. Med., 2007). Lung cells in IPF patients show telomere shortening, DNA damage response (DDR) at telomeres and markers of cellular senescence, which increase with disease severity (Lee, S. et al., Nat. Commun, 2021). In independent cohorts, shorter telomere length is a robust independent predictor of disease progression, response to therapy and death, and is associated with a shorter time to allograft dysfunction following lung transplant (Stuart, B. D. et al. Lancet Respir. Med., 2014). Genetic or pharmacological clearance of senescent cells improves lung function and health in a mouse model of bleomycin-induced IPF (Schafer, M. J. et al. Nat. Commun., 2017).
Mouse models indicate that telomere dysfunction can recapitulate IPF features. Mice with short telomeres induced by genetic knockout of telomerase components show telomere damage, inflammation and fibrosis in lungs (Chen et al, 2015). Telomere dysfunction, specifically induced in alveolar epithelial type II cells through the deletion of the telomere proteins TRF1 or TRF2, leads to impaired regeneration, inflammation and fibrosis (Povedano et al, 2015; Alder et al, 2015). Consistently, re-expression of telomerase using adeno-associated vectors (AAVs) in alveolar epithelial type II cells led to reduction in telomeric DDR, decreased inflammation and improved lung function in telomerase-mutant and wild-type aged mice (Povedano et al, 2018; Piñeiro-Hermida et al, 2020).
Hematopoietic and Immune Manifestations Associated with Telomere Dysfunction (Dyskeratosis Congenita, Aplastic Anemia, Altered Myeloid Progenitor Differentiation, and Bone Marrow Failure)
Telomere dysfunction can lead to various hematological manifestations, including Dyskeratosis Congenita (DC). DC was the first disorder to be linked to a pathogenic variant in telomere maintenance genes (Roka, 2023). DC is a multisystem syndrome characterized by a wide range of clinical manifestations affecting different organs. Clinical symptoms usually appear during childhood but there is significant variability among patients regarding age of onset and severity of disease. The major and most common clinical manifestations are the mucocutaneous triad of nail dystrophy, abnormal skin pigmentation and leukoplakia and the bone marrow failure (BMF)/immune deficiency, which affects up to 80% of DC patients by the age of 30, representing also the main cause of mortality (˜60-70%). Other recognized manifestations of the disease are pulmonary fibrosis (PF) and cancer, representing the cause of mortality for 10-15% and 10% of the patients, respectively (Carvalho, 2022; Tummala, 2022).
In patients with significative peripheral cytopenia, androgen therapy is the first line of treatment, but with major adverse effects, including virilization and liver toxicity, often requiring treatment discontinuation (Calado, 2017). Patients who do not respond to androgens are good candidates for allogenic Hematopoietic Stem Cell Transplantation (HSCT), which to date is the only curative treatment for the hematopoietic and immunologic abnormalities. Even so, there is a significant mortality associated with HSCT for DC patients, due to pulmonary and vascular complications, the toxicity of drugs used in the conditioning regimen, the risk of infections and rejection and the difficulty in finding a compatible donor. In addition, HSCT does not cure the extra-hematopoietic manifestations of the disease, such as the pulmonary fibrosis, for which there are no standard treatments (Savage, 2022). Moreover, short telomeres are associated with reduced response rate to standard therapies and more frequent relapses (Young, 2018; Narita, 2015; Sakaguchi, 2014). Therefore, new approaches targeting all the manifestations of the disease are needed.
Mutations in at least 18 different genes with a role in telomere maintenance have been identified so far. The most commonly mutated are DKC1 (25%), TINF2 (12%), TERC (5%), TERT (4%) and RTEL1 (3%), with all mode of inheritance represented (X-linked recessive for DKC1, autosomal dominant for TINF2, TERC and TERT, and autosomal recessive for RTEL1). Each of the remaining characterized mutations represents less than 1% of DC patients and 25% of patients still have uncharacterized mutations (Tummala, 2022).
As a consequence, DC patients show shortened and dysfunctional telomeres and telomere dysfunction is a commonly accepted hallmark and driver of age-associated organs decline, including features common to DC, such as pulmonary fibrosis and hematopoietic dysfunction. Telomeric DNA is made of TTAGGG repeats and telomeres represent the ending portion of mammalian chromosomes. Telomere dysfunction can be caused by telomere shortening below a critical length (telomeres shorten at each cycle of cell division): when critically-shortened telomeres become unable to bind enough telomere-capping proteins that prevent them from being recognized as DNA damage, they initiate a DNA-damage response (DDR) leading to cellular senescence (Rossiello, 2022).
DDR at telomeres seems to be an important actor involved in the physiopathology of DC. It was shown that pathogenic mutations in murine Dkc1 cause a growth disadvantage following an enhanced DNA damage response at telomeres (Gu, 2008). Moreover, analysis of cells from DC patients of several genetic subtypes showed increased damage foci at telomeres in lymphocytes and an increase in the basal level of DNA damage in fibroblasts (Kirwan, 2011).
Brain ageing is characterized by a progressive decline in memory and cognition and is recognized as the greatest risk factor for neurodegenerative diseases. DNA damage accumulation and the activation of the DNA damage response (DDR) have been consistently shown to correlate with ageing and proposed to be one of its main causative mechanisms (Schumacher et al, 202, Stavgiannoudaki et al, 2024). It is conceivable that augmented and pathological DDR, due to impaired DNA repair system and accumulation of persistent DDR may play a relevant role in neurodegeneration.
Indeed, the ability to maintain genomic integrity is probably most important for post-mitotic cells that are long-lived; these include neurons which are for the most part irreplaceable and have to rely on the integrity of their genetic material for the lifetime of an organism.
Indeed, DNA damage in human samples from patient and in mouse model of neurodegeneration has been described (Myung et al, 2008; Simpson et al, 2014), and patient affected by genetic mutation in DNA repair system and mouse model harboring mutation in DNA repair genes show neurological phenotype as the most prominent (Mckinnon et al 2010, Biton et al, 2008, Sepe et al, 2014), suggesting that DNA damage accumulation can triggers dysfunction in the brain leading to neurodegeneration. Several evidences suggest that telomeres might be sources of persistent DNA damage in ageing even without overt telomere shortening in various mice tissues including the brain (Fumagalli et al., 2012; Hewitt et al., 2012), because the DNA damage at telomeres is highly stable and not readily repaired (Fumagalli et al., 2012; Hewitt et al., 2012).
Among neurodegeneration, Alzheimer's disease (AD) is the most common one in adults over age 65. AD is a progressive neurodegeneration with progressive memory loss and cognitive dysfunction. Indeed, clinical features of AD include impairment of recent memory, language disturbances, and alterations of abstract reasoning, concentration and thought sequencing (executive function). AD is pathologically characterized by amyloid-β protein (AB) accumulation, plaque formation, neurofibrillary tangles, and the hyperphosphorylation and polyubiquitination of the tau protein (Masters et al 2015). Current therapeutic approaches aim to alleviate symptoms but often come with significant side effects (Robinson and Keating 2006). Therefore, exploring alternative molecular pathways for more specific treatments is crucial for AD.
WO2013/167744 relates to small RNAs molecules (DDRNAs) produced at a site of DNA damage and having the specific sequence of the damaged locus. The existence and generation of DDRNAs from telomeres in cells exhibiting ALT, such as ALT cancer cells, or in EBV-infected ALT cells or in cells from premature ageing conditions is not described neither it is suggested DDRNA inhibition as therapeutic rationale.
WO2014092609 relates to a method for influencing the proliferative status of cells using specific G-chain oligonucleotide sequences of human telomeric DNA.
US2013065950 discloses compounds comprising an oligonucleotide moiety covalently linked to a lipid moiety. The oligonucleotide moiety comprises a sequence that is complementary to the RNA component of human telomerase. The compounds inhibit telomerase activity in cells.
WO97/38013 does not refer specifically to a disease (in particular a cancer) characterized by alternative lengthening of telomeres (ALT) neither to a non-cancer condition characterized by dysfunctional telomeres. In addition, it relates to inhibition of telomerase. By contrast, the oligonucleotides of the present invention are not active on telomerase-positive cancer cells.
WO 2006/107949 refers to a method of treating oxidative stress disorders. The present invention relates to the specific treatment of ALT cancer cells and non-cancer conditions characterized by dysfunctional telomeres.
CN1936011 relates to a polycation liposome telomere enzyme antisense oligonucleotide compound that is made up of polycation liposome and antisense oligonucleotide.
WO2006028160 relates to phosphorothioate oligonucleotide conjugate which has high inhibitory activity against the telomerase contained in a human leukemic cell extract and which gives a stable duplex hybrid with a complementary DNA.
US2006183704 relates to methods for treating hyperproliferative disorders, including cancers, by administering to an affected mammal an effective amount of a composition comprising pTT or a composition comprising one or more oligonucleotides which share at least 50% nucleotide sequence identity with the human telomere overhang repeat.
WO01/74342 relates to methods of treatment or prevention of hyperproliferative diseases or precancerous conditions affecting epithelial cells, such as psoriasis, vitiligo, atopic dermatitis, or hyperproliferative or UV-responsive dermatoses, hyperproliferative or allergically mediated diseases of other epithelia and methods for reducing photoaging, or oxidative stress or for prophylaxis against or reduction in the likelihood of the development of skin cancer.
WO96/23508 discloses a method of inhibiting proliferation of cancer and other immortal type cellular disease states. The method includes introduction of synthetic oligonucleotides which mimic telomere motifs.
Sandra Sampl et al. (Proceedings: AACR Annual Meeting 2014; Apr. 5-9, 2014; Abstract 2743, San Diego, CA), indicate that RNA transcripts from telomeres called TERRA were identified to block telomerase activity (TA) potentially via direct binding to TERC.
TERRA are constitutive single-stranded non-coding RNA, from around 100 bases up to at least 9 kilobases in length (Azzalin et al., 2007). They are transcribed starting from a promoter located in the subtelomeric region, thus they carry both subtelomeric and the G-rich telomeric sequences. Differently, DDRNAs are short RNA (at least 6 or 8 nucleotides long or between 10 and 50 nucleotides long, around 22 nucleotides long) that can potentially form double-stranded RNAs. They are generated by processing of precursor transcripts, which are transcribed from both telomeric strands, generating a G-rich and a C-rich telomeric repeats-containing RNA molecules, starting at the very end of telomeres or within telomeric repeats.
Therefore there is still the need for treatment of diseases characterized by alternative lengthening of telomeres and non-cancer conditions associated with telomere dysfunction.
The present invention is based on the discovery that upon telomeric DNA damage or dysfunction non-coding RNAs (named tDDRNAs) accumulate. These are RNA transcripts synthesized using a dysfunctional telomere as a template for transcription of a long RNA precursor, which can be then processed by Dicer and/or Drosha into shorter non-coding RNAs (tDDRNAs).
In the present invention it was surprisingly found that inhibitors of generation and/or synthesis and/or functions of tDDRNAs and their precursors also inhibit DDR activation and thus may be applied for the treatment of ALT-associated conditions and conditions associated with telomeric DNA damage or dysfunction.
Antisense oligonucleotides (ASOs), for instance in the form of locked nucleic acids (LNA) complementary to tDDRNAs and/or their precursors were synthetized, together with LNA with unrelated sequences as negative control. It was observed that telomeric ASOs can specifically inhibit DDR activation, inhibiting both signaling and ensuing DNA repair.
With a sequence-specific ASO, results showing decreased cell proliferation specifically in ALT cells were obtained.
Telomeric DDR activation in ALT tumors may be targeted by sequence-specific ASOs, thus impairing telomere maintenance and reducing proliferation. The inventors synthesized LNA ASOs with the sequence complementary to telomeric RNAs generated upon telomere DNA damage or dysfunction. They observed that transfection of an ASO oligonucleotide carrying a specific telomeric sequence was able to strongly suppress the proliferation of ALT positive cell lines (i.e. osteosarcoma U-2 OS and G292 cell lines, glioblastoma GBM14 cell lines, fibroblasts WI38 VA13 and SW26) while the same amount of ASO with a different sequence not targeting telomeric RNA and not targeting any human sequence had no significant effect. Importantly, none of these ASOs has an impact on normal human fibroblasts (of mesenchymal origin like osteosarcomas) proliferation or on the proliferation of telomerase-positive cancer cells, as tested in parallel, suggesting that this approach is specific for ALT tumors and indicates that this treatment will not be toxic in living animals and human patients. At this stage, they have:
Furthermore, ageing is associated with DDR activation especially at telomeres (d'Adda di Fagagna et al., 2003; Fumagalli et al., 2012; Herbig et al., 2006; Herbig et al., 2004; Hewitt et al., 2012). The Hutchinson-Gilford progeria syndrome (HGPS) (Pollex and Hegele, 2004) is one example of a progeria syndrome caused by a mutant form of lamin A, also called progerin. Progeria means premature ageing. HGPS progeric phenotypes can be suppressed by the expression of telomerase, by decreasing progerin-induced DNA-damage signaling (Kudlow et al., 2008). This indicates that the progeric phenotype of HGPS cells (and of HGPS patients by extension) is caused by dysfunctional telomeres causing DDR. Indeed DDR is found at the telomeres in HGPS cells (Benson et al., 2010; Chojnowski et al., 2015). The fish Danio rerio (zebrafish) is a simple vertebrate model, suitable for ageing studies. Indeed it has been shown that telomerase mutation accelerates physiological ageing by speeding up telomere shortening and consequent telomere dysfunction and DDR activation (Henriques et al., 2013). Specifically telomerase-mutant fish are characterized by shorter lifespan, tissue atrophy and decreased fertility. They represent a model of non-cancer condition associated with telomere dysfunction.
By using LNA ASOs with the sequence complementary to tDDRNAs and their precursors generated upon telomere DNA damage or dysfunction the inventors prevented cellular senescence in progerin-expressing cells. Moreover they extended the lifespan of a mouse model for HGPS and of telomerase-mutant zebrafish, suggesting that they could suppress ageing-related phenotypes.
In the present invention, the inhibition of DDR through the inhibition of DDRNAs and/or their precursors have the great advantage to be sequence specific, thus minimizing the possibility of adverse side effects in normal cells due for instance by DDR inhibition away from telomeres.
Further, in the context of cancer therapeutics, these inhibitors may synergize with telomerase inhibitors in order to prevent the potential emergence of clones of cancer cells using ALT mechanism of telomere maintenance and thus resistant to telomerase inhibitions.
In fact, existing anticancer therapies are effective anticancer treatments that act by damaging the DNA of or inhibiting DDR in cancer cells. However the DNA damaging activity or the DDR inhibition of most of them is not sequence specific. The oligonucleotides of the present invention impair DDR in a sequence-specific manner, then they may also confer sequence specificity to existing DNA damaging treatments thus enhancing efficacy.
Therefore, the present invention provides an oligonucleotide comprising one of the following sequence:
Preferably the oligonucleotide or a fragment or a variant thereof comprises one of the following sequence: (TTAGGG)n, (TAGGGT)n, (AGGGTT)n, (GGGTTA)n, (GGTTAG)n or (GTTAGG)n wherein 1<n<1000, preferably 1<n<500, preferably 1<n<200, preferably 1<n<100, preferably 1<n<50, preferably 1<n<20, preferably 1<n<10, preferably 1<n<5.
Preferably said oligonucleotide is complementary to the sequence of an RNA, said RNA being a RNA transcript synthesized using a specific dysfunctional telomeric DNA as a template for transcription or a fragment of said RNA transcript, said fragment (DDRNA) being generated by processing by Dicer and/or Drosha.
Preferably the disease is cancer or an Epstein-Bar virus infection. Still preferably the disease is ALT-positive cancer.
Yet preferably the cancer is selected from the group consisting of: soft tissue sarcoma, preferably chondrosarcoma, undifferentiated pleomorphic sarcomas including malignant fibrous histiocytoma, leiomyosarcoma, epithelioid sarcoma, liposarcoma, fibrosarcoma and variants, angiosarcoma and neurofibroma, central nervous system cancer, preferably grade 2 diffuse astrocytoma; grade 3 anaplastic astrocytoma; grade 4 paediatric glioblastoma multiforme, oligodendroglioma, anaplastic medulloblastoma, grade 1 pilocytic astrocytoma, nonanaplastic medulloblastoma, meningioma, schwannoma, urinary bladder cancer, in particular small cell carcinoma and invasive urothelial carcinoma, adrenal gland or peripheral nervous system cancer, in particular ganglioneurobalstoma, neuroblastoma and pheochromocytoma, neuroendocrine neoplasms such as paraganglioma and carcinoid tumour, kidney cancer, in particular chromophobe carcinoma, sarcomatoid carcinoma and clear cell and papillary carcinomas, lung and pleural cancer, in particular malignant mesothelioma, large cell carcinoma and small cell carcinoma, skin cancer such as malignant melanoma, liver cancer such as hepatocellular carcinoma, testis cancer such as non seminomatous germ cell tumor, breast cancer, in particular lobular carcinoma; ductal carcinoma and medullary carcinoma, uterus cancer such as serous endometrial carcinoma, squamous carcinoma of cervix, ovary cancer, in particular clear cell carcinoma, endometrioid carcinoma, Gall bladder cancer such as adenocarcinoma, oesophagus cancer.
In a further aspect the invention provides an oligonucleotide comprising one of the following sequence:
Preferably the non-cancer condition associated with telomere dysfunction is selected from the group consisting of: Hutchinson-Gilford progeria syndrome (HGPS), Werner's syndrome, Bloom's syndrome, ataxia telangiectasia, familial IPF, sporadic IPF, aplastic anaemia, autosomal-dominant dyskeratosis congenital, Familial MDS-AML, de novo dyskeratosis congenita, X-linked recessive dyskeratosis congenita, Hoyeraal-Hreiderasson syndrome, Revesz syndrome, Autosomal-recessive dyskeratosis congenita, Coats plus syndrome, condition caused by mutations or inactivation of any one of TRF1, POT1, TPP1, TINF2, RAP1 or TRF2, impaired regeneration upon partial hepatectomy, liver fibrosis, liver Chronic inflammation, liver cirrhosis, pulmonary fibrosis, altered myeloid progenitor differentiation, bone marrow failure, chronic obstructive pulmonary disease (COPD), neurological disorders including Alzheimer's Disease, osteoporosis, atherosclerosis, heart disease, Duchenne muscular dystrophy, Type 2 diabetes, impaired fertility, impaired wound healing, arthritis, cataracts, age-related macular degeneration, ageing.
Preferably the oligonucleotide is a locked nucleic acid (LNA)-modified oligonucleotide or a 2′-O-Methyl-modified oligonucleotide.
LNA is generally considered to be an RNA mimic in which the ribose sugar moiety is locked by an oxymethylene bridge connecting the C (2′)- and C (4′)-atoms which conformationally restricts LNA monomers into an N-type sugar puckering (Veedu R et al. 2010). LNA is a molecule which contains at least one nucleotide bearing the LNA modification. Preferably the LNA contains, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 nucleotides bearing the LNA modification.
2′-O-Methyl-modified oligonucleotide is a molecule which contains at least one nucleotide bearing the 2′-O-Methyl modification. Preferably the 2′-O-Methyl-modified oligonucleotide contains, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 nucleotides bearing the 2′-O-Methyl modification.
In a further aspect the invention provides a pharmaceutical composition comprising at least one oligonucleotide as defined above and pharmaceutically acceptable carriers for use in the treatment and/or prevention of a disease characterized by alternative lengthening of telomeres or for use in the treatment and/or prevention of a non-cancer condition associated with telomere dysfunction.
Preferably the pharmaceutical composition further comprises at least another therapeutic agent, preferably the other therapeutic agent is selected from the group of: anti-tumoral agent, anti-pain agent, anti-emetic agent (such as aprepitant, fosaprepitant, Dolasetron, granisetron, ondansetron, palonosetron, tropisetron, or ramosetron, Dexamethasone).
Preferably the other therapeutic agent is selected from the group consisting of: ATR inhibitor, DDR inhibitor, HR inhibitor, molecule that specifically target telomeres, preferably G-quadruplexes interacting molecules, molecule that cause DNA damage generation specifically at telomeres.
In a further aspect the invention provides a method to identify a subject to be treated with the oligonucleotide as defined above or with the pharmaceutical composition as defined above comprising detecting the presence and/or measuring the amount of an RNA, said RNA being a RNA transcript synthesized using a specific dysfunctional telomeric DNA as a template for transcription or a fragment of said RNA transcript, said fragment (DDRNA) being generated by processing by Dicer and/or Drosha wherein said subject is affected by a disease characterized by alternative lengthening of telomeres or affected by a non-cancer condition associated with telomere dysfunction.
In the present invention DDRNAs are non-coding RNAs. They are RNA transcripts synthesized using a specific damaged and/or dysfunctional telomeric DNA as a template for transcription of a long RNA precursor which can be then processed by Dicer and/or Drosha into shorter non-coding RNAs (DDRNAs or tDDRNAs).
DDRNAs originate at the locus and carry the sequence of the damaged locus. When chemically synthesized or generated in vitro by DICER and/or DROSHA cleavage of transcripts spanning the locus, DDRNAs promote DDR activation at the DNA damage site in RNase A-treated cells even in the absence of other mammalian RNAs.
DDRNAs act differently from microRNAs and canonical RNAi mechanisms because as shown in (Francia et al., 2012):
DDRNAs are small RNAs, with the potential to form double-stranded pairs, they are generated by processing by DICER and/or DROSHA of a sequence-specific RNA transcript synthesized upon transcription of a damaged DNA locus. DDRNAs are small RNAs of a length between 10 and 50 nucleotides. For example of a length between 17 and 32 nucleotides. For example of a length between 20 and 25 nucleotides. For example of a length between 21 and 23 nucleotides.
Said DDRNAs function by favoring the sequence-specific accumulation of DDR factors at specific sites of DNA damage and promote DDR activation (i.e. comprising, but not limiting to, DNA damage signalling, such as through protein phosphorylation events, and DNA damage repair, such as homologous recombination).
DDRNA precursors are RNA molecules longer than DDRNAs (at least 25 bases long, preferably at least 30 bases long, preferably at least 50 bases long, preferably at least 100 bases long, preferably at least 150 bases long, preferably at least 200 bases long, preferably at least 250 bases long, preferably at least 300 bases long), transcribed upon DNA damage, using damaged DNA as a template. They are processed by DROSHA and/or DICER to generate DDRNAs. Telomeric DDRNA precursors are multiples of telomeric DDRNAs.
In the present invention a fragment of SEQ ID No. 1 to SEQ ID No. 6 is a functional fragment that has the same therapeutic activity as said sequences. The fragment corresponds to the oligonucleotides of the present invention that are truncated by one or more nucleotides on the 5′end, the 3′end, or both the 5′end and the 3′end, so long as at least two contiguous nucleotides of the untruncated oligonucleotide remain. Preferably the truncated oligonucleotides have 2, 3, 4 or 5 contiguous nucleotides found in the untruncated oligonucleotides.
Comprised in the present invention are also oligonucleotides comprising the above sequences (SEQ ID No. 1 to 6) that are repeated 1 or more times, for instance oligonucleotides comprising one of the following sequence: (TTAGGG)n, (TAGGGT)n, (AGGGTT)n, (GGGTTA)n, (GGTTAG)n or (GTTAGG)n wherein 1<n<1000, preferably 1<n<500, preferably 1<n<200, preferably 1<n<100, preferably 1<n<50, preferably 1<n<20, preferably 1<n<10, preferably 1<n<5.
The oligonucleotides may be also 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25 etc. . . . nucleotide long. They are not necessarily multiple of 6 nucleotides.
In the present invention the variant of SEQ ID No. 1 to 6 are oligonucleotides in which 1, 2, 3, 4 or 5 nucleotides are substituted with a different nucleotide. The variants have at least 50% identity with SEQ ID No. 1 to 6, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% identity. The variant has the same therapeutic activity as the oligonucleotide.
Preferred telomeric hexanucleotide variants include: TCAGGG, TTCGGG, GTAGGG, TGAGGG, TTGGGG, TAAGGG, ATAGGG, CTAGGG, TTTGGG, TTAAGGG and their complementary sequences (
The oligonucleotide of the present invention, fragment thereof or variant thereof is complementary to the sequence of DDRNAs and/or their precursors, thereby inhibiting DDRNAs and/or their precursors function.
Preferably the oligonucleotide is a LNA molecule or a 2′-O-Methyl modified oligonucleotide.
These oligonucleotides comprise but are not limiting to locked nucleic acids (LNA), phosphorothioate modified oligonucleotides, phosphorothioate modified locked nucleic acids, 2′-O-methoxyethyl modified oligonucleotides, 2′ O-Methyl modified oligonucleotides, 2O-[2-(N-Methylcarbamoyl)ethyl] ribonucleosides, methylphosphonates, morpholino oligonucleotides, LNA-DNA-LNA gapmer oligonucleotides, mixmers, Chimeric 2′-O-methyl RNA-DNA gapmer, N3′-P5′ Phosphoroamidate, 2′-fluoro-arabino nucleic acid, Phosphoroamidate Morpholino, Cyclohexene nucleic acid, Tricyclo-DNA, Peptide nucleic acid, Unlocked nucleic acid, Hexitol nucleic acid, Boranophosphate oligonucleotides, Phosphoroamidate oligonucleotides, preferably said modified oligonucleotide is phosphorothioated, and/or oligonucleotides expressed by plasmid-encoded genes delivered by different means (comprising but not limited to plasmid transfection, viral infection).
In the present invention a disease characterized by alternative lengthening of telomeres is a disease characterized by the presence of cells that maintain telomeres despite lacking telomerase activity and/or showing one or more features listed below.
In particular, alternative lengthening of telomeres may be identified/measured by at least one of the following feature:
These features may be measured/identified by known methods in the art, for instance as reported in (Henson and Reddel, 2010) (included by reference herein).
In the present invention a non-cancer condition associated with telomere dysfunction is a disease or condition or syndrome characterized by telomeres engaging the components of the DDR machinery. In the present invention “telomere dysfunction” or “dysfunctional telomere” is a telomere with damaged telomeric DNA and/or a critically short telomeric DNA and/or uncapped telomeric DNA and/or deprotected telomeric DNA and/or accelerated telomere loss and/or any instance where DDR signal is active at a telomere.
Telomere dysfunction has a causal role in a number of degenerative disorders. Their manifestation encompass common disease states such as Hutchinson-Gilford progeria syndrome (HGPS), Werner's syndrome, Bloom's syndrome, ataxia telangiectasia, familial IPF, sporadic IPF, aplastic anaemia, autosomal-dominant dyskeratosis congenital, Familial MDS-AML, de novo dyskeratosis congenita, X-linked recessive dyskeratosis congenita, Hoyeraal-Hreiderasson syndrome, Revesz syndrome, Autosomal-recessive dyskeratosis congenita, Coats plus syndrome, condition caused by mutations or inactivation of any one of TRF1, POT1, TPP1, TINF2, RAP1 or TRF2, impaired regeneration upon partial hepatectomy, liver fibrosis, liver Chronic inflammation, liver cirrhosis, pulmonary fibrosis, altered myeloid progenitor differentiation, bone marrow failure, chronic obstructive pulmonary disease (COPD), neurological disorders including Alzheimer's Disease, osteoporosis, atherosclerosis, heart disease, Duchenne muscular dystrophy, Type 2 diabetes, impaired fertility, impaired wound healing, arthritis, cataracts, age-related macular degeneration, ageing. In example, accelerated telomere loss, i.e a telomere dysfunction, has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis (Rudolph et al. 2000).
Although these disorders seem to be clinically diverse, collectively they comprise a spectrum of syndromes characterized by short or damaged and more broadly dysfunctional telomeres, and telomere dysfunction is also a key feature of ageing and age-related diseases (see (Armanios and Blackburn, 2012; Gray et al., 2015; Opresko and Shay, 2016; Wang et al., 2015; Xi et al., 2013; Satyanarayana et al, 2003; Rudolph et al. 2000)).
Further telomere dysfunction may be caused by mutations or inactivation, in particular of TRF1 (official name TERF1, gene ID 7013), POT1 (gene ID 25913), TPP1 (official name ACD, gene ID 65057), TINF2 (gene ID 26277), RAP1 (official name TERF2IP, gene ID 54386) or TRF2 (official name TERF2, gene ID 7014).
In addition, TRF2KO models cause telomere dysfunction and recapitulate the non-cancer conditions associated with telomere dysfunction.
Telomere dysfunction may be identified/measured by at least one of the following feature or method: indirect immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, tDDRNA detection.
More specifically, the invention pertains to the use of polynucleotides, oligonucleotides, deoxynucleotides, dinucleotides, or dinucleotide dimers, or similar compounds, for the inhibition of cell proliferation or inhibition of DNA repair. As used herein, inhibition of cell proliferation includes complete abrogation of cell division, partial inhibition of cell division and transient inhibition of cell division, cell death, apoptosis, necrosis, cellular senescence, cell differentiation, mitotic catastrophe as measured by standard tests in the art and as described in the Examples. The invention also pertains to the prevention and/or treatment of diseases characterized by ALT, including, but not limited to, cancer and pre-cancerous conditions, wherein the disease affects cells of any organ and any embryonic origin. Metastatic ALT tumors and cancers that have regrown or relapsed after treatment, as well as primary tumors, can be treated by the methods of the invention.
In one embodiment, the compositions of the present invention comprise oligonucleotides approximately 2-200 bases in length, which can be administered to a mammal (e. g., human) in an appropriate vehicle. In another embodiment, the oligonucleotides are about 5 to about 100 nucleotides in length. In still another embodiment, the oligonucleotides are about 5 to about 50 nucleotides in length. In yet another embodiment, the DNA oligonucleotides are about 8-30 nucleotides in length. Preferably they are 8-21 nucleotide in length, still preferably 8-16 nucleotide in length.
Any suitable method of administering the oligonucleotide of the present invention to the organism, such that the oligonucleotide contacts and/or enters the cells or tissues of interest, is reasonably expected to be effective. The effects can be optimized using routine optimization protocols.
The oligonucleotide, deoxynucleotides of the present invention can be obtained from any appropriate source, or can be synthetically produced.
DNA fragments, oligonucleotides, deoxynucleotides, dinucleotides or dinucleotide dimers, may be applied to the skin and can be administered alone, or in combination with physiologically acceptable carriers, including solvents, perfumes or colorants, stabilizers, sunscreens or other ingredients, for medical or cosmetic use. They can be administered in a vehicle, such as water, saline, or in another appropriate delivery vehicle. The delivery vehicle can be any appropriate vehicle, which delivers the oligonucleotides, deoxynucleotides, dinucleotides, or dinucleotide dimers. In one embodiment, the concentration range of oligonucleotide can be from 0.1 nM to 500 M, preferably the in vitro range is between 0.2 nM and 300 μM, preferably the in vitro range is between 0.5 nM to 200 μM. Preferred in vivo range is 0.1-500 mg/kg, preferably in vivo range is 1-50 mg/kg.
To allow access of the active ingredients of the composition to deeper-lying skin cells, vehicles which improve penetration through outer layers of the skin, e. g., the stratum corneum, are useful. Vehicle constituents for this purpose include, but are not limited to, ethanol, isopropanol, diethylene glycol ethers such as diethylene glycol monoethyl ether, azone (1-dodecylazacycloheptan-2-one), oleic acid, linoleic acid, propylene glycol, hypertonic concentrations of glycerol, lactic acid, glycolic acid, citric acid, and malic acid. In one embodiment, propylene glycol is used as a delivery vehicle. In a preferred embodiment, a mixture of propylene glycol:ethanol:isopropyl myristate (1:2.7:1) containing 3% benzylsulfonic acid and 5% oleyl alcohol is used.
In another embodiment, a liposome preparation can be used. The liposome preparation can comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell. For example, liposomes such as those described in U.S. Pat. No. 5,077,211 of Yarosh, U.S. Pat. No. 4,621,023 of Redziniak et al. or U.S. Pat. No. 4,508,703 of Redziniak et al. can be used. The compositions of the invention intended to target skin conditions can be administered before, during, or after exposure of the skin of the mammal to UV or agents causing oxidative damage. Other suitable formulations can employ niosomes. Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
Other suitable delivery methods intended primarily for skin include use of a hydrogel formulation, comprising an aqueous or aqueous-alcoholic medium and a gelling agent in addition to the oligonucleotide(s). Suitable gelling agents include methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, carbomer (carbopol), hypan, polyacrylate, and glycerol polyacrylate.
In one embodiment, the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or composition comprising one or more of the foregoing, is applied topically to the skin surface. In other embodiments, the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or composition comprising one or more of the foregoing, is delivered to other cells or tissues of the body such as epithelial cells. Cells of tissue that is recognized to have a lesser barrier to entry of such substances than does the skin can be treated, e. g., orally to the oral cavity; by aerosol to the respiratory epithelium; by instillation to the bladder epithelium; by instillation or suppository to intestinal (epithelium) or by other topical or surface application means to other cells or tissues in the body, including eye drops, nose drops and application using angioplasty, for example. Furthermore, the oligonucleotides of the present invention can be administered intravenously or injected directly into the tissue of interest intracutaneously, subcutaneously, intramuscularly or intraperitoneally. In addition, for the treatment of blood cells, the compounds of the present invention can be administered intravenously or during extracorporeal circulation of the cells, such as through a photophoresis device, for example. As demonstrated herein, all that is needed is contacting the cells of interest with the oligonucleotide compositions of the present invention.
The oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, or composition comprising one or more of the foregoing, is administered to (introduced into or contacted with) the cells of interest in an appropriate manner.
The “cells of interest” as used herein are those cells which may become affected or are affected by a disease characterized by ALT or by dysfunctional telomeres.
The oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, agent that promotes differentiation, or composition comprising one or more of the foregoing, is applied at an appropriate time, in an effective amount. The “appropriate time” will vary, depending on the type and molecular weight of the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or other agent employed, the condition to be treated or prevented, the results sought, and the individual patient.
An “effective amount” as used herein, is a quantity or concentration sufficient to achieve a measurable desired result. The effective amount will depend on the type and molecular weight of the oligonucleotides, deoxynucleotides, dinucleotides, dinucleotide dimers, or agent employed, the condition to be treated or prevented, the results sought, and the individual patient. For example, for the treatment or prevention of ALT hyperproliferative disease, cancerous, or pre-cancerous conditions, the effective amount is the amount necessary to reduce or relieve any one of the symptoms of the disease, to reduce the volume, area or number of cells affected by the disease, to prevent the formation of affected areas, or to reduce the rate of growth of the cells affected by the hyperproliferative disorder.
As demonstrated herein, the inhibitors of the present invention are active in vitro and in vivo in their unmodified form, e. g., sequences of unmodified oligonucleotides linked by phosphodiester bonds. As used herein, the terms “oligonucleotide”, “dinucleotide,” etc., refer to molecules having ribose and/or deoxyribose as the sugar, and having phosphodiester linkages (“phosphate backbone”) as occur naturally, unless a different linkage or backbone is specified.
Oligonucleotides are relatively short polynucleotides. Polynucleotides are linear polymers of nucleotide monomers in which the nucleotides are linked by phosphodiester bonds between the 3′ position of one nucleotide and the 5′ position of the adjacent nucleotide. Unless otherwise indicated, the “oligonucleotides” of the invention as described herein have a phosphodiester backbone.
To enhance delivery through the skin, the oligonucleotides of the invention may be modified so as to either mask or reduce their negative charges or otherwise alter their chemical characteristics. This may be accomplished, for example, by preparing ammonium salts of the oligonucleotides using readily available reagents and methods well known in the art. Preferred ammonium salts of the oligonucleotides include trimethyl-, triethyl-, tributyl-, tetramethyl-, tetraethyl-, and tetrabutyl-ammonium salts. Ammonium and other positively charged groups can be convalently bonded to the oligonucleotide to facilitate its transport across the stratum comeum, using an enzymatically degradable linkage that releases the oligonucleotide upon arrival inside the cells of the viable layers of the epidermis.
Another method for reducing or masking the negative charge of the oligonucleotides includes adding a polyoxyethylene spacer to the 5′phosphate groups of the oligonucleotides and/or the internal phosphates of the oligonucleotides using methods and reagents well known in the art. This, in effect, adds a 6- or 12-carbon modifier (linker) to the phosphate that reduces the net negative charge by +1 and makes the oligonucleotides less hydrophilic.
Further negative charge reduction is achieved by adding a phosphoroamidite to the end of the polyoxyethylene linker, thereby providing an additional neutralizing positive charge. The phosphodiester backbone of the oligonucleotides of the present invention can also be modified or synthesized to reduce the negative charge. A preferred method involves the use of methyl phosphonic acids (or chiral-methylphosphonates), whereby one of the negatively charged oxygen atoms in the phosphate is replaced with a methyl group. These oligonucleotides are similar to oligonucleotides having phosphorothioate linkages which comprise a sulfate instead of a methyl group and which are also within the scope of the present invention.
The oligonucleotides of the present invention can also take the form of peptide nucleic acids (PNAs) in which the bases of the nucleotides are connected to each other via a peptide backbone.
Other modifications of the oligonucleotides such as those described, for example, in U.S. Pat. Nos. 6,537,973 and 6,506,735 as well as in (Stenvang et al., 2012) (all of which are incorporated herein by reference for all of the oligonucleotide modifications described therein) and others will be readily apparent to those skilled in the art.
The oligonucleotides can also be “chimeric” oligonucleotides which are synthesized to have a combination of two or more chemically distinct backbone linkages, one being phosphodiester. In one embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 3′ end. In one embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 5′ end. In another embodiment chimeric oligonucleotides are with one or more phosphodiester linkages at the 3′ and 5′ ends.
The oligonucleotide or oligonucleotides to be used to treat and/or prevent a disease characterized by alternative lengthening of telomeres, such as cancer or an Epstein-Bar virus infection, in particular soft tissue sarcoma, preferably chondrosarcoma, undifferentiated pleomorphic sarcomas including malignant fibrous histiocytoma, leiomyosarcoma, epithelioid sarcoma, liposarcoma, fibrosarcoma and variants, angiosarcoma and neurofibroma, central nervous system cancer, preferably grade 2 diffuse astrocytoma; grade 3 anaplastic astrocytoma; grade 4 paediatric glioblastoma multiforme, oligodendroglioma, anaplastic medulloblastoma, grade 1 pilocytic astrocytoma, nonanaplastic medulloblastoma, meningioma, schwannoma, urinary bladder cancer, in particular small cell carcinoma and invasive urothelial carcinoma, adrenal gland or peripheral nervous system cancer, in particular ganglioneurobalstoma, neuroblastoma and pheochromocytoma, neuroendocrine neoplasms such as paraganglioma and carcinoid tumour, kidney cancer, in particular chromophobe carcinoma, sarcomatoid carcinoma and clear cell and papillary carcinomas, lung and pleural cancer, in particular malignant mesothelioma, large cell carcinoma and small cell carcinoma, skin cancer such as malignant melanoma, liver cancer such as hepatocellular carcinoma, testis cancer such as non seminomatous germ cell tumor, breast cancer, in particular lobular carcinoma; ductal carcinoma and medullary carcinoma, uterus cancer such as serous endometrial carcinoma, squamous carcinoma of cervix, ovary cancer, in particular clear cell carcinoma, endometrioid carcinoma, gall bladder cancer such as adenocarcinoma, oesophagus cancer or to treat and/or prevent for use in the treatment and/or prevention of a non-cancer condition associated with telomere dysfunction, in particular Hutchinson-Gilford progeria syndrome (HGPS), Werner's syndrome, Bloom's syndrome, ataxia telangiectasia, familial IPF, sporadic IPF, aplastic anaemia, autosomal-dominant dyskeratosis congenital, Familial MDS-AML, de novo dyskeratosis congenita, X-linked recessive dyskeratosis congenita, Hoyeraal-Hreiderasson syndrome, Revesz syndrome, Autosomal-recessive dyskeratosis congenita, Coats plus syndrome, condition caused by mutations or inactivation of any one of TRF1, POT1, TPP1, TINF2, RAP1 or TRF2 impaired regeneration upon partial hepatectomy, liver fibrosis, liver Chronic inflammation, liver cirrhosis, pulmonary fibrosis, altered myeloid progenitor differentiation, bone marrow failure, chronic obstructive pulmonary disease (COPD), neurological disorders including Alzheimer's Disease, osteoporosis, atherosclerosis, heart disease, Duchenne muscular dystrophy, Type 2 diabetes, impaired fertility, impaired wound healing, arthritis, cataracts, age-related macular degeneration, ageing.
For instance such diseases are described in (Durant, 2012) (enclosed by reference herein).
The oligonucleotides can be used in a composition in combination with a pharmaceutically or physiologically acceptable carrier. Such a composition may also contain in addition, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Cationic lipids such as DOTAP [N-(2, 3-dioleoyloxy) propyl]-N, N, N-trimethylammonium salts may be used with oligonucleotides to enhance stability. Oligonucleotides may be complexed with PLGA/PLA copolymers, chitosan or fumaric acid/sebacic acid copolymers for improved bioavailability {where PLGA is [poly (lactide-co-glycolide)]; PLA is poly (L-lactide)}. The terms “pharmaceutically acceptable” and “physiologically acceptable” mean a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
A composition to be used as an antiproliferative agent for ALT disease may further contain other agents which either enhance the activity of the oligonucleotide(s) or complement its activity or use in treatment, such as chemotherapeutic or radioactive agents. Such additional factors and/or agents may be included in the composition to produce a synergistic effect with the oligonucleotide(s), or to minimize side effects. Additionally, administration of the composition of the present invention may be administered concurrently with other therapies, e. g., administered in conjunction with a chemotherapy or radiation therapy regimen. The oligonucleotides as described herein can be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation, chemotherapy, or immunotherapy, combined with oligonucleotide therapy, and then oligonucleotides may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor. The oligonucleotides can be used in combination with telomerase inhibitors, to prevent the expansion of telomerase-negative, ALT-positive clones.
Preferably the other therapeutic agent is selected from the group consisting of: ATR inhibitor, DDR inhibitor, HR inhibitor, molecule that specifically targets and/or causes DNA damage generation specifically at telomeres, preferably G-quadruplexes interacting molecules.
In the present invention an ATR inhibitor is a small molecule compound able to inhibit the kinase activity of ATR, comprising but not limited to VE-821 (Vertex Pharmaceuticals), VE-822 (Vertex Pharmaceuticals), AZ20 (AstraZeneca), AZD6738 (AstraZeneca) (as described in (Flynn et al., 2015; Weber and Ryan, 2015) (all references are incorporated by reference).
A DDR inhibitor is any compound or experimental approach able to impair or inhibit the cellular process known as DNA damage response (DDR), comprising but not limited to: Caffeine, Wortmannin, KU-55933, KU-60019, KU-559403, Schisandrin B, NU6027, NVP-BEZ235, (as described in (Begg et al., 2011; Kelley et al., 2014; Weber and Ryan, 2015), all references are incorporated by reference).
A HR inhibitor is any compound or experimental approach able to impair or inhibit the cellular process known as DNA repair by homologous recombination (HR), comprising but not limited to: Iniparib (SAR240550, BSI-201; Sanofi-Aventis), Olaparib (AZD2281, KU-0069436; AstraZeneca), Niraparib (Tesaro), Rucaparib (CO-338, AG-014699, PF-01367338; Pfizer), Veliparib (ABT-888; Abbott), AZD2461 (AstraZeneca), BMN673 (BioMarin Pharmaceutical), CEP-9722 (Cephalon), E7016 (Esai), INO-1001 (Inotek Pharmaceuticals), MK-4827 (Merck), Methoxyamine (Sigma Aldrich), RI-1, IBR2, B02, Halenaquinone (described in (Feng et al., 2015; Kelley et al., 2014; Ward et al., 2015), all references are incorporated by reference).
A molecule that specifically targets and/or causes DNA damage generation at telomeres is any compound or experimental approach which specifically or preferentially interacts with telomeres, inducing DNA damage within telomeric DNA and/or activation or inhibition of DDR signalling and/or DNA repair, comprising but not limited to: G-quadruplex-binding ligands (e.g. BRACO-19, Telomestatin, RHPS4, Quarfloxin, TMPyP4, AS1410), topoisomerase inhibitors, cisplatin, hydroxyurea, (as described in (Lu et al., 2013; Muller and Rodriguez, 2014; Neidle, 2010; Salvati et al., 2015; Sissi and Palumbo, 2014), all references are incorporated by reference).
Other molecules that can be used in combination with the oligonucleotides are: Abitrexate (Methotrexate Injection), Abraxane (Paclitaxel Injection), Adcetris (Brentuximab Vedotin Injection), Adriamycin (Doxorubicin), Adrucil Injection (5-FU (fluorouracil)), Afinitor (Everolimus), Afinitor Disperz (Everolimus), Alimta (PEMETREXED), Alkeran Injection (Melphalan Injection), Alkeran Tablets (Melphalan), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab Injection), Avastin (Bevacizumab), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Busulfex Injection (Busulfan Injection), Campath (Alemtuzumab), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), Clolar (Clofarabine Injection), Cometriq (Cabozantinib), Cosmegen (Dactinomycin), CytosarU (Cytarabine), Cytoxan (Cytoxan), Cytoxan Injection (Cyclophosphamide Injection), Dacogen (Decitabine), DaunoXome (Daunorubicin Lipid Complex Injection), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex Injection), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex Injection), Droxia (Hydroxyurea), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Erbitux (Cetuximab), Erivedge (Vismodegib), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Injection), Eulexin (Flutamide), Fareston (Toremifene), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix Injection), Fludara (Fludarabine), Folex (Methotrexate Injection), Folotyn (Pralatrexate Injection), FUDR (FUDR (floxuridine)), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine wafer), Halaven (Eribulin Injection), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Ifex (Ifosfamide), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin Injection), Ixempra (Ixabepilone Injection), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel Injection), Kadcyla (Ado-trastuzumab Emtansine), Kyprolis (Carfilzomib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lupron (Leuprolide), Lupron Depot (Leuprolide), Lupron DepotPED (Leuprolide), Lysodren (Mitotane), Marqibo Kit (Vincristine Lipid Complex Injection), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mesnex (Mesna), Mesnex (Mesna Injection), Metastron (Strontium-89 Chloride), Mexate (Methotrexate Injection), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Neosar Injection (Cyclophosphamide Injection), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (nilutamide)), Nipent (Pentostatin), Nolvadex (Tamoxifen), Novantrone (Mitoxantrone), Oncaspar (Pegaspargase), Oncovin (Vincristine), Ontak (Denileukin Diftitox), Onxol (Paclitaxel Injection), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab Injection), Platinol (Cisplatin), Platinol (Cisplatin Injection), PlatinolAQ (Cisplatin), PlatinolAQ (Cisplatin Injection), Pomalyst (Pomalidomide), Prednisone Intensol (Prednisone), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Sandostatin (Octreotide), Sandostatin LAR Depot (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Sterapred (Prednisone), Sterapred DS (Prednisone), Stivarga (Regorafenib), Supprelin LA (Histrelin Implant), Sutent (Sunitinib), Sylatron (Peginterferon Alfa-2b Injection (Sylatron)), Synribo (Omacetaxine Injection), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel Injection), Taxotere (Docetaxel), Temodar (Temozolomide), Temodar (Temozolomide Injection), Tepadina (Thiotepa), Thalomid (Thalidomide), TheraCys BCG (BCG), Thioplex (Thiotepa), TICE BCG (BCG), Toposar (Etoposide Injection), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin Injection), Trexall (Methotrexate), Trisenox (Arsenic trioxide), Tykerb (lapatinib), Valstar (Valrubicin Intravesical), Vantas (Histrelin Implant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Vepesid (Etoposide), Vepesid (Etoposide Injection), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin Injection), Xalkori (Crizotinib), Xeloda (Capecitabine), Xtandi (Enzalutamide), Yervoy (Ipilimumab Injection), Zaltrap (Ziv-aflibercept Injection), Zanosar (Streptozocin), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid), Zortress (Everolimus), Zytiga (Abiraterone), Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab/MK-3475, pidilizumab and AMP-224 targeting PD-1; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.
Radiotherapy means the use of radiation, usually X-rays, to treat illness. X-rays were discovered in 1895 and since then radiation has been used in medicine for diagnosis and investigation (X-rays) and treatment (radiotherapy). Radiotherapy may be from outside the body as external radiotherapy, using X-rays, cobalt irradiation, electrons, and more rarely other particles such as protons. It may also be from within the body as internal radiotherapy, which uses radioactive metals or liquids (isotopes) to treat cancer.
Still further aspects include combining the oligonucleotides described herein with other anticancer therapies for synergistic or additive benefit.
Existing anticancer therapies are effective anticancer treatments that act by damaging the DNA or inhibiting DDR of cancer cells. However the DNA damaging activity of most of them is not sequence specific. The oligonucleotides of the present invention impair DDR in a sequence-specific manner, then they may confer sequence specificity to existing DNA damaging treatments thus enhancing efficacy.
The schedule of treatment with the combinations can foresee that the oligonucleotide is administered concomitantly, before and/or after any of the “partner” therapeutic agent identified above.
Combination therapies can be utilized for advanced stage of disease but also, prospectively, in the adjuvant and neo-adjuvant setting.
In the present invention “dysfunctional telomeric DNA” is a damaged telomeric DNA and/or a critically short telomeric DNA and/or uncapped telomeric DNA and/or deprotected telomeric DNA and/or any instance where DDR signal is active at a telomere.
The compositions of the present invention can be in the form of a liposome in which oligonucleotide(s) of the present invention are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Pharmaceutical compositions can be made containing oligonucleotides to be used in antiproliferative therapy. Administration of such pharmaceutical compositions can be carried out in a variety of conventional ways known to those of ordinary skill in the art, such as oral ingestion, inhalation, for example, of an aerosol, topical or transdermal application, or intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e. g., intravenous, intraspinal, subcutaneous or intramuscular) route, or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. The route of administration can be determined according to the site of the tumor, growth or lesion to be targeted. To deliver a composition comprising an effective amount of one or more oligonucleotides to the site of a growth or tumor, direct injection into the site can be used. Alternatively, for accessible mucosal sites, ballistic delivery, by coating the oligonucleotides onto beads of micrometer diameter, or by intraoral jet injection device, can be used. Viral vectors for the delivery of DNA in gene therapy have been the subject of investigation for a number of years. Retrovirus, adenovirus, adeno-associated virus, vaccina virus and plant-specific viruses can be used as systems to package and deliver oligonucleotides for the treatment of cancer or other growths. Adeno-associated virus vectors have been developed that cannot replicate, but retain the ability to infect cells. An advantage is low immunogenicity, allowing repeated administration. Delivery systems have been reviewed, for example, in (Page and Cudmore, 2001). Studies carried out using oligonucleotides on the theory of their inhibiting the function of a target nucleic acid (antisense oligonucleotides), most of these studies carried out with phosphorothioate oligonucleotides, have found effective methods of delivery to target cells. Antisense oligonucleotides in clinical trials have been administered in saline solutions without special delivery vehicles (reviewed in (Hogrefe, 1999)). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to oligonucleotide(s) of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
Use of timed release or sustained release delivery systems are also included in the invention. Such systems are highly desirable in situations where surgery is difficult or impossible, e. g., patients debilitated by age or the disease course itself, or where the risk-benefit analysis dictates control over cure. One method is to use an implantable pump to deliver measured doses of the formulation over a period of time, for example, at the site of a tumor. A sustained-release matrix can be used as a method of delivery of a pharmaceutical composition comprising oligonucleotides, especially for local treatment of a growth or tumor. It is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly (ortho) esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamin acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid). The amount of oligonucleotide of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. For a human patient, the attending physician will decide the dose of oligonucleotide of the present invention with which to treat each individual patient. Initially, the attending physician can administer low doses and observe the patient's response. Larger doses may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. The duration of therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
The present invention also provides a kit comprising at least one oligonucleotide as defined above or pharmaceutical composition comprising the same. The kit may contain written instructions. The oligonucleotide may be in a separate container.
In the present invention the method to identify a subject to be treated with the oligonucleotide as defined above or with the pharmaceutical composition as defined above may further comprise comparing the measured amount of DDRNAs or tDDRNAs to a control amount. The control amount may be the amount measured in a healthy subject, the control amount may be the amount measured in a subject not affected by an ALT-disease or not affected by a non-cancer condition associated with telomere dysfunction, the control amount may be the amount measured in the same subject before or after therapeutic intervention.
The invention will be illustrated by means of non-limiting examples in reference to the following figures.
SEM of 3-5 biological replicates. C. Bar graph represents cell viability as determined by CellTiter-Glo and shown as a percentage of surviving cells compared to the vehicle. Data presented as mean of 7 biological replicates±SEM. D. Bar graph represents the quantification of the ratio between the mRNA levels of BAX over BCL2 relative to vehicle as measured by RT-qPCR. Data presented as mean of 3 biological replicates±SEM. *p<0.05, **p<0.01, ***p<0.001.
Cultured cells: MEFs CRE-ER TRF2fl/fl and MEFs CRE-ER TRF2fl/+ (Celli and de Lange, 2005) were grown in DMEM supplemented with 10% fetal bovine serum and 1% glutamine; for CRE activation and TRF2 knock out induction, cells were treated with 600 nM of 4 hydroxytamoxifen for 24 hours and analysed 24 hours later. U-2 OS cells (ATCC) were grown in DMEM supplemented with 10% fetal bovine serum and 1% glutamine. Saos-2 cells (ATCC) were grown in McCoy's 5A+Glutamax supplemented with 15% fetal bovine serum. WI-38 and WI-38 VA-13 (ATCC) were grown in MEM+Glutamax supplemented with 10% fetal bovine serum, 10 mM non-essential amino acids and 1 mM sodium pyruvate. The telomerase-positive cell line SW39 (ALT negative) and the ALT-positive cell line SW26 (Bechter et al., 2003) were grown in in a 4:1 mixture of Dulbecco modified Eagle medium-medium 199 supplemented with 10% defined supplemented bovine calf serum. BJ hTERT were obtained by retroviral infection of BJ cells (ATCC) with a human telomerase expressing plasmid, and were grown in MEM+Glutamax supplemented with 10% fetal bovine serum, 10 mM non-essential amino acids and 1 mM sodium pyruvate. BJ ELR (Hahn et al., 1999) were grown in DMEM: M199 4:1 supplemented with 10% fetal bovine serum, 1% glutamine, 1 mM sodium pyruvate and 25 mM HEPES. GBM14 (ALT positive) were grown in DMEM/F12 with GlutaMAX supplemented with 2% of B27, 5 μg/ml Heparin, 20 ng/ml of bFGF and 20 ng/ml of EGF. G-292 (ATCC) were grown in McCoy's 5A+Glutamax supplemented with 10% fetal bovine serum and 1% glutamine.
Transfection: LNA were boiled at 90° C. for 5 minutes and chilled in ice for 5 minutes before transfection with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions at the indicated final concentration. Mock-transfected cells were treated with RNAiMAX only.
Growth curves: At each indicated time point cells were counted in triplicate with a Coulter Counter (Beckman) or with the In Vitro Toxicology Assay Kit, Resazurin based (Sigma), which allows a spectrophotometric measurement of metabolic activity of living cells, in accordance with the manufacturer's instructions.
Immunofluorescence: Cells were fixed with 1:1 methanol/acetone solution for 2 minutes at room temperature. After blocking, cells were stained with anti PML (Santa Cruz) primary antibody for 1 hour at room temperature, washed and incubated with conjugated anti mouse secondary antibodies for 40 minutes at room temperature. Nuclei were stained with DAPI (1 μg/ml). Confocal sections were obtained with a Leica TCS SP2 AOBS confocal laser microscope by acquisition of optical z sections at different levels along the optical axis and number of APBs per cell was counted by CellProfiler software.
RNA isolation: Total cellular RNA was extracted using mirVana™ miRNA Isolation kit (Life Technologies) according to the manufacturer's instructions.
qPCR of small RNA: cDNA synthesis and RT-PCR were performed using the miScript PCR system (Qiagen). RNA was fractionated by running 5 μg of total RNA on a polyacrylamide denaturing gel. RNA species shorter than 40 nucleotides were gel extracted and cDNA was synthesized using the miScript II RT kit with HiSpec buffer. Reactions were incubated at 37° C. for 60 min followed by a heat-inactivation step for 5 min at 95° C. cDNA was analysed using the miScript SYBR Green PCR Master Mix, miScript Universal Primer, mir29b primer (TAGCACCATTTGAAATCAGTGTT) SEQ ID No. 7, Spike-In primer (CGAATTCCACAAATTGTTATCC) SEQ ID No. 8 to monitor efficiency of RNA extraction from gel and telomere sequence-containing primers (TAGGGTTAGGGTTAGGGT, SEQ ID No. 9, CCCTAACCCTAACCCTAA SEQ ID No. 10).
Strand specific qPCR: Total RNA coming from mirVana™ miRNA Isolation Kit was used. Samples were treated with DNase I (Thermo Scientific) to remove any potential residual genomic DNA contamination. 1000 ng of total RNA were reverse-transcribed using the Superscript First Strand cDNA synthesis kit (Invitrogen) with strand-specific primers. Primers for reverse-transcription used: RPPOrev for the detection of the housekeeping Rplp0 mRNA; teloCrev for the detection of G-rich-stranded telomeric precursor; teloGrev for the detection of C-rich-stranded telomeric precursor.
RT-qPCR was performed using Roche SYBR green. For each RT-qPCR reaction, 50 ng of cDNA were used. To amplify telomeric repeats the inventors adapted a technique described in (Cawthon, 2002), which allows the generation of a fixed-length amplification product. Primers for qPCR used: RPPOfwd and RPPOrev for the detection of the housekeeping Rplp0 mRNA; teloF and teloR for the detection of telomeric precursors.
Below, the list of primers (5′-3′ orientation) used for strand-specific RT-qPCR:
Targeted sequencing of small RNA. Two linkers were ligated to the two ends of the RNA molecules in the sample to be analysed. The 3′ end of the starting RNA was ligated to a monoadenylated DNA linker by a T4 RNA ligase 2 truncated enzyme (NEB) incubated for 1 hour at 25° C. The 5′ RNA linker was then ligated by a T4 RNA ligase 1 (NEB) to the target RNA at 20° C. for 1 hour, after removing the 5′ cap structure by Tobacco Acid Pyrophosphatase (Epicentre), incubated at 37° C. for 1 hour. Linkers enabled cDNA synthesis using PrimeScript RT-PCR Kit (Takara). The reverse transcription reaction was incubated at 44° C. for 1 hour. Subsequent PCR amplification using Phusion® High-Fidelity DNA Polymerase (NEB) was carried out as follows: 98° C. 2 minutes; 22 cycles of: 98° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds; 72° C. for 5 minutes; hold at 4° C. To capture the amplified cDNA targets, complementary RNA baits containing biotin-labeled nucleotides were used. These RNA baits were produced by using AMbion MAXIscript T7 In Vitro Transcription kit (Life Technologies) and Biotin RNA labelling Mix (Roche). A T7-promoter-containing dsDNA was incubated at 37° C. for 1 hour to allow for in-vitro transcription. RNA baits and cDNA targets were incubated at 37° C. for 48 hours in the presence of SUPERase-inhibitor (Life Technologies) and of the following blocking agents: Human Cot-1 (Life Technologies), UltraPure™ Salmon Sperm DNA Solution (Thermo Scientific) and a 200 μM Customized Block. The hybrid RNA-cDNA molecules were captured by Dynabeads® MyOne™ Streptavidin C1 (Life Technologies) beads, while non-targeted cDNAs were washed away. Captured cDNAs were then barcoded by PCR with Script Index PCR primers (Illumina) and sequenced by a MiSeq (Illumina) sequencer. Oligonucleotide sequences (5′-3′ orientation) were:
Western blot: Cells were collected in lysis buffer TEB150 (50 mM Hepes, 150 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 0.5% Triton, 10% glycerin), and flash frozen in liquid nitrogen until sample preparation for western blot. To lyse, cells were thawed on ice, spun for 15 minutes at +4° C. and 13200 RPM. The protein-containing supernatant was saved, and cell debris trashed. Proteins were quantified using the Bradford assay. After transfer, membranes were probed with an anti-caspase-3 antibody (Cell Signaling 9661), an anti-PARP antibody (Serotec), and an anti-tubulin antibody (Millipore). For Lamin A/C, Progerin, and Tubulin, cells were collected in Laemmli 1× buffer and stored at −80° C. To lyse, cells were passed through a syringe and boiled at 95° C. for 5 minutes. Proteins were quantified using the Lowry assay. After transfer, membranes were probed with anti-Lamin antibody (Santa Cruz Biotech sc-6215), recognizing both Lamin isoforms A and C, and Progerin and anti-Tubulin antibody (Millipore).
FACS analysis: For cell cycle analysis, the medium supernatant was saved and spun down with trypsinized cells, fixed in 75% ethanol and stained with Propidium Iodine (PI, Sigma, 50 μg/ml) and RNase A (Sigma, 250 g/ml) solution in 1×PBS. For caspase-3 cleavage analysis by FACS, the medium supernatant was saved and spun down with trypsinized cells, fixed in 1% formamide on ice for 20 minutes, stored in 75% ethanol, stained with Caspase-3 antibody (Cell Signaling 9661) and subsequently conjugated to the fluorophore FITC (anti rabbit FITC, ImmunoJackson), and stained with PI/RNase A solution. Samples were analyzed on a BD Facs CantoII, using a 488 nm laser and 530/30 filter for FITC, and 670 nm laser and 585/42 filter for PI. Acquisition was performed with the software BDFacsDIVA v6.1.1, and analysis was done using software ModfitLT3.0. For Sub G-1 and caspase positive cells, at least 500 events were analyzed per sample. For cell cycle, at least 8000 events were analyzed per sample.
Naked Delivery: Oligonucleotides in PBS were added straight to plated cells, while mock-treated cells were given only PBS. A constant amount of PBS was used per condition in each experiment.
Retroviral infection: BJ cells were transduced with a retroviral vector expressing either wild type LaminA or mutant Progerin gene and selected with puromycin.
In vivo treatment of the G292 xenografts: CD-1 nude male mice, from Charles River Italy were maintained in cages using steam autoclaved (sterile) bedding, y-radiated diet and acidified mineral water.
10×106 G-292 cells were injected subcutaneously into the left flank of nude male mice at day 0. Animals were examined regularly for the appearance of tumors. When tumors had reached a volume of 90 to 220 mm3, mice were randomized and assigned to treatment groups, with a target of 7 mice per group. When treatment starts the mean tumor volume was about 0.14 cm3. Treatments were administered intraperitoneally at a dose of 15 mg/kg at day 13, 17, 21, 25 for PBS and Tiny anti TeloC, day 13 and 17 for PS anti TeloC.
All procedures adopted for housing and handling of animals were in strict compliance with Italian and European guidelines for Laboratory Animal Welfare. Body weight at the day of tumor implant: g. 25-38.
In vivo treatment of the Tert mutant zebrafish: Heterozygous telomerase mutant zebrafish (Tert+/−) were crossed and the eggs were injected at one cell stage with 0.5 ng/μl of PS LNA into the yolk. The injected fish were raised to adulthood in the nursery and then genotyped by fin clips to identify the homozygous mutant fish (Generation 1 tert−/−), which were crossed and their offspring (Generation 2) was analyzed for survival rate and phenotype.
In vivo treatment of HGPS mice: HGPS mice expressing the progerin in epidermal keratinocytes (McKenna et al, Aging cell 2014) were intraperitoneally injected with the PS LNA oligonucleotides at a concentration of 15 mg/Kg every 3-4 days starting at embryonic day 17.5.
LNA sequences: The LNA oligonucleotides were produced by Exiqon.
LNA with a phosphate backbone (
LNA with a fully phosphorothioate backbone (LNA-PS) (
Tiny (8-mer) LNA with a fully phosphorothioate backbone (
2′-O-Methyl oligonucleotides (
Statistical analysis: Results are shown as mean plus or minus standard deviation or standard error of the mean. P value was calculated by Student's two-tailed t-test, Chi-square test, Mann Whitney test or Mantel-Cox test, as appropriate.
The inventors measured the levels of DDRNAs generated at damaged telomeres by qPCR. To do so, they used mouse embryonic fibroblasts (MEFs) in which telomeres can be deprotected by knocking out the telomere binding protein TRF2 (Celli and de Lange, 2005). This leads to the activation of DDR at virtually all telomeres. This is an accepted model of telomere dysfunction. It has already been shown that, when the DNA is damaged, small RNA molecules named DDRNAs are generated at that specific damaged locus, and that they carry the same sequence of the damaged DNA (Francia et al., 2012); thus the inventors reasoned that two different sets of molecules of DDRNAs could be generated upon telomere deprotection, a set deriving from the transcription of the G-rich telomeric DNA strand (TeloC DDRNA) and a set deriving from the transcription of the C-rich telomeric DNA strand (TeloG DDRNA;
In cells with deprotected telomeres, both TeloG and TeloC DDRNA precursor transcripts were strongly induced when compared to control cells (
ALT-positive cells display a strong chronic DDR activation at the telomeres (Cesare and Reddel, 2010). The inventors discovered that this correlates with higher levels of tDDRNAs, as detected by RT-qPCR, in ALT-positive WI-38 VA-13, compared with their parental cell line, WI-38 human fibroblasts, and in the ALT-positive SW26 compared with the telomerase-positive SW39 fetal lung fibroblasts, which are cell lines immortalized from the same fibroblast cell line, IMR90, resulting in different telomere maintenance mechanisms (Bechter et al., 2003) (
ALT-positive cells rely on the homologous recombination mechanism to maintain their telomeres (Cesare and Reddel, 2010); thus the inventors tested whether they are hypersensitive to DDRNA inhibition.
The inventors transfected an ALT cell line, U-2 OS human osteosarcoma cells, with locked nucleic acid (LNA) (Veedu and Wengel, 2010) molecules targeting either the G or the C-rich telomeric transcripts (anti TeloG and anti TeloC, respectively), or a control LNA, and the inventors monitored the cell growth for ten days. The growth of the cells transfected with the control and the anti TeloG LNA did not differ from the mock-treated cells; differently, the anti TeloC LNA significantly impaired U-2 OS growth (
The effect is specific for ALT cells, since the anti TeloC LNA did not have a significant impact on growth rate of telomerase-expressing human fibroblast cells, either transformed (BJ ELR), or normal (BJ hTERT) (
To monitor the impact of LNA treatment on ALT biomarkers, the inventors evaluated the presence of ALT-associated PML bodies (APBs), nuclear structures containing recombination factors and telomeric DNA specific of ALT cells (Henson and Reddel, 2010). By an indirect immunostaining against the PML protein, the inventors observed a decrease of APBs in U-2 OS cells treated with the anti TeloC LNA, compared with the control or the anti TeloG LNA (
In order to extend their observations, the inventors transfected the ALT-positive cell lines U-2 OS, Saos-2 and WI-38 VA-13 with anti TeloG and anti TeloC LNA. The anti TeloC LNA significantly inhibited the cell growth in all three cell lines tested (
So called “tiny/short LNA oligonucleotides”, which are at least 6 nt, preferably at least 8 nt long fully LNA oligonucleotides, have been shown to target specifically their complementary RNA target, both in vitro and in vivo (Obad et al., 2011). The inventors designed tiny/short LNA oligonucleotides with a phosphorothioate backbone, named “tiny anti TeloC” and “tiny anti TeloG”, which are 8 nucleotides in length and target the telomeric transcripts (
U-2 OS cells transfected with tiny anti TeloC oligonucleotide grew significantly less than mock transfected cells or cells transfected with a control or tiny anti TeloG oligonucleotide (
By using a 3-fold higher amount of the tiny LNA oligos (60 nM) proportional to their shorter length and thus to their ability to match a telomeric repeats RNA, the inventors observed an inhibitory effect on ALT cell proliferation of the tiny anti TeloC LNA, similar to the PS anti TeloC LNA at 20 nM concentration (
Anti TeloC LNA-mediated growth impairment, by both PS LNA and Tiny LNA, was accompanied by induction of apoptosis, evidenced by caspase-3 cleavage, and increased sub G-1 cells indicative of cell death, as shown by FACS analysis, as well as by caspase-3 and PARP-1 cleavage, as shown by western blot analysis, (
To further prove the specificity of anti TeloC LNAs, the inventors tested the effect of LNA transfection on paired cell lines SW26 (ALT) and SW39 (telomerase-positive), described previously. The anti TeloC LNAs inhibited growth of ALT cells much more than the matched non-ALT controls (
As transfection of cells can be achieved in vitro, but not in vivo, it is important to test the ability of cells to uptake “naked” LNA, with no transfection agent. This process is termed “gymnotic delivery,” and is believed to be a better predictor of the effectiveness of a treatment in vivo (Stein et al., 2010). Thus, the inventors sought to determine the efficacy of naked delivery of LNAs in multiple ALT cell lines.
Naked Delivery of LNAs was effective in U-2 OS cells, both PS anti TeloC LNA and Tiny anti TeloC LNA inhibited cell growth (
In order to demonstrate that the efficacy of the phosphorothioate backbone oligonucleotides was not restricted to the osteosarcoma cell lines, the inventors tested them on another cell type, the ALT-positive glioblastoma cell line GBM14 (Heaphy et al., 2011) by naked delivery. Only the PS anti TeloC and tiny anti TeloC LNA significantly reduced cell growth compared to untreated cells (
Another ALT-positive cell line, G-292, is capable of growing as a xenograft in mice (Lauvrak et al., 2013). The inventors first determined that this cell line was also affected by only anti TeloC LNA and not controls (
To test the efficacy of the ASO treatment on the growth of ALT-positive tumors in vivo, G-292 cells were injected into the flank of nude mice, until they formed a detectable tumor mass. Tumor-bearing mice were treated intraperitoneally with ASOs. Both the PS anti TeloC and the tiny anti TeloC reduced the tumor growth compared to the vehicle (PBS)-injected mice (
The inventors tested the efficacy and the specificity of another class of ASOs, 2′-O-Methyl (2′-O-Me) ASOs with a phosphorothioate backbone. Only U-2 OS transfected with 2′-O-Me anti-TeloC grew significantly less than mock-transfected cells, while control and anti-TeloG ASO did not affect cell growth (
Non-Cancer Conditions Associated with Telomere Dysfunction
Genetic inactivation of telomerase functions in fish Danio rerio (zebrafish) induces telomere dysfunction and a number of pathological events recapitulating ageing in an accelerated form (Anchelin et al., 2013; Carneiro et al., 2016). Therefore this is an established faithful vertebrate model of telomere dysfunction, in particular of physiological ageing. Second-generation telomerase-mutant zebrafish animals were studied because of a stronger phenotype compared to first generation zebrafish. The animals are characterized by morphological defects and shorter lifespan and animals die few days after birth. PS LNA were injected in one-cell embryos from first generation telomerase mutant fish, which were crossed to obtain a second generation. The fish born from individuals treated with either anti TeloC or anti TeloG showed a significant reduction of the morphological defects associated with premature ageing (
The inventors infected normal human fibroblasts with a vector expressing the mutated form of the Lamin A gene, also known as progerin (Gonzalo et al., 2016) or the wild-type Lamin A gene as control. This gene is mutated in the HGPS patients and its expression leads to a slow down of the cell growth and to premature senescence, recapitulating the premature ageing phenotype observed in telomere syndrome such as the HGPS patients. The progerin expression has been shown to cause telomere dysfunction (Chojnowski et al., 2015). The inventors monitored the cell growth of these cells upon PS LNA transfection. In the presence of either PS anti TeloC or PS anti TeloG, the progerin-expressing cells grew more than cells mock transfected or transfected with the PS control LNA, as monitored by BrdU incorporation and expression of the proliferation marker KI67 (
A HGPS mouse model, which expresses the progerin in epidermal keratinocytes shows epidermal hyperplasia, severe skin abnormalities, hair thinning, marked hyperkeratosis, moderate fibrosis of the dermis, infiltration by inflammatory cells and they die within the first two postnatal weeks (McKenna et al., 2014). The inventors treated the progerin-expressing and wild-type mice with PS anti TeloG LNA once during pregnancy, injecting the pregnant females, and every 3 days after birth. The treatment with the anti TeloG significantly prolonged the lifespan of progeric mice, compared with untreated animals (
tDDR Control by tASOs
Dysfunctional telomeres are actively transcribed to generate telomeric non-coding RNAs (tncRNAs) which are essential to activate and recruit DNA Damage Response (DDR) factors at damaged telomeres, thus fueling DNA damage signaling and causing cellular senescence initiation and maintenance. Sequence-specific telomeric antisense oligonucleotides (tASOs) target these tncRNAs and effectively inhibit telomeric DDR (tDDR) activation in cultured cells and ameliorate age-related phenotypes in mouse models (Rossiello, 2017; Aguado, 2019; Sepe 2022). In particular, DDR inhibition through tASOs treatment significantly improved the progeroid pathological phenotype of a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), characterized by telomere dysfunction, genomic instability and premature entry into cellular senescence (Aguado, 2019), validating tASOs as a therapeutic agent for HGPS and for other diseases caused by telomeric dysfunction.
Patients with idiopathic pulmonary fibrosis often have germiline mutations in genes involved in telomere maintenance, including telomerase. Therefore, we used a mouse model lacking the RNA component of telomerase (Terc−/−), which, at late generations, shows telomere shortening and recapitulates several features of IPF (Piñeiro-Hermida et al, Journal of cell biol 2020).
To evaluate the impact of tASOs treatment on the DNA damage response (DDR) and cellular senescence in vivo, in a model of IPF, third generation (G3) Terc−/− mice were treated with tASOs (anti-teloG or anti-teloC) or an ASO not complementary to any sequence in the mammalian genome, used as a negative control (control ASO) and sacrificed at 12 months of age, 9-month after the treatment period (
Quantitative analysis of hematoxylin-eosin-stained lung sections revealed a significantly higher pathological score based on different pathological parameters (inter-alveolar septal thickening, interstitial cellularity increase, interstitial inflammatory infiltration, intra-alveolar inflammatory infiltration and erythrocyte extravasation), in control ASO-treated G3 Terc−/− mice compared to wild-type controls, indicating increased tissue damage and inflammation (
Masson's Trichrome staining, highlighting collagen fibers deposition, indicated a significant increase in fibrosis in the lungs of control ASO-treated G3 Terc−/− mice compared to wild-type controls, as quantified by density analysis (
RNA-sequencing analysis revealed that tASOs treatment restored the differentially expressed cellular pathways, which were dysregulated in the G3 Terc−/− mice, including DNA repair, inflammation, and immune activation (
It has been reported that telomere dysfunction in alveolar type II (ATII) stem cells, can drive lung fibrosis (Povedano, Cell reports 2015). To investigate the impact of tASO treatment on this specific cell population, which play a crucial role in lung function and regeneration. Lung sections were stained for prosurfactant protein C (pro-SPC) to identify ATII cells. Immunohistochemical and immunofluorescence analyses revealed a significant increase in the percentage of gH2AX-, 53BP1-, and p16-positive ATII cells in the lungs of control ASO-treated G3 Terc−/− mice compared to wild-type controls (
To assess the impact of tASOs treatment on lung pathology in older animals with more advanced fibrosis, 10-month-old G3 Terc−/− mice were treated with the indicated ASOs for 4 weeks and subsequently sacrificed at 20 months of age, 9 months after the treatment period (
Overall, these results indicate that tASOs treatment effectively reduces DNA damage response, cellular senescence, pathological alterations, fibrosis, and immune cell infiltration, in a mouse model for IPF.
Hematopoietic and Immune Manifestations Associated with Telomere Dysfunction (Dyskeratosis Congenita, Aplastic Anemia, Altered Myeloid Progenitor Differentiation, and Bone Marrow Failure)
To test the efficacy of tASOs treatment in hematopoietic manifestations associated with telomere dysfunction, we first used the telomerase-deficient (Terc−/−) mouse model. This model lacks the RNA component of telomerase (Terc), the enzyme that synthesizes telomeric repeats de novo, crucial for the maintenance of telomeric length in mammalian cells and one of the genes most commonly mutated in DC. Telomeric length in Terc−/− mice shortens by approximately 5 kb in subsequent generations, with phenotypic abnormalities appearing after the 3rd generation (G3) (Blasco. 1997). Terc−/− mice are characterized by abnormalities of the hematopoietic and immune system, including a reduction in immune system reactivity (immunosenescence), decreased lymphocytes numbers and increased neutrophil count and decreased long term renewal of hematopoietic stem cells (Blasco, 1997; Samper, 2002; Allsopp, 2003; Ju, 2007; Chen, 2015).
We first evaluated the impact of tASOs treatment on the DNA damage response (DDR) in G3 Terc−/− mice treated with tASOs (anti-teloG or anti-teloC) or a control ASO and sacrificed 2 months after the treatment period (
We then wanted to better characterize the hematopoietic/immune phenotype of G3 Terc−/− mice. Flow cytometric analysis of splenocytes from young (3 to 5-month-old) and adult (12-month-old) mice revealed decreased percentages of B cells in adult but not young G3 Terc−/− mice (
Since G3 Terc−/− mice show altered percentages of immune cells in adulthood, we investigating if inhibiting tDDR by tASOs could affect these parameters. We collected BM and spleens from adult G3 Terc−/− mice treated with tASOs or a control ASO and sacrificed 9 months after the treatment period (
Quantitative analysis of hematoxylin-eosin-stained BM (
We wanted to study the effects of tASOs treatment on HSPCs function. Since a diminished HSPCs fitness characterize G3 Terc−/− mice already at a young age (
Finally, the best way to check how effectively HSPCs are functioning within the body is the BM transplant. We therefore performed a competitive BM transplant experiment (
Overall, these results indicate that tASOs treatment effectively reduces DDR and pathological alterations in both BM and spleen of G3 Terc−/− mice, restoring at the same time the normal percentages of immune cells. Most importantly, tASO treatment effectively impact HSPCs function, by improving their fitness in vitro and their reconstitution ability in vivo.
Here, we investigated the nexus between DDR and AD by evaluating the possibility that the telomeric DNA damage response might contribute to the toxicity induced by Aβ oligomer accumulation. We used mouse primary neurons and human cortical neurons derived from iPSCs exposed to Aβ oligomers, which trigger several pathological features observed in AD.
The intraneuronal Aβ oligomers accumulation has been showed to be an important pathogenic trigger for onset of cognitive deficits in this mouse model (Billing et al, 2005) preceding the amyloid plaque formation. Thus, in order to determine whether pathological elevation of Aβ causes neuronal telomeric DNA damage, we exposed hippocampal mouse primary neurons to Aβ42 oligomers, which are the major toxic forms during the amyloid plaque formation process, hallmark of AD pathology (Sengupta et al, 2016) In addition, they have been shown to induce DNA damage (Suberbielle et al, 2013; Jung et al, 2016). We observed that Aβ42 oligomers trigger the DDR in the mouse primary neurons, as demonstrated by the focal accumulation and activation of DDR factors such as gH2AX, 53BP1 and pATM (
We next explored whether the DNA damage at telomeres was more abundant due to its propensity to persist. Interestingly, our findings have revealed a distinct accumulation of these markers at telomeres, as demonstrated by the co-localization study between telomeres and gH2AX (
Animals and treatments: Experiments involving animals have been done in accordance with the Italian Laws (D.lgs. 26/2014), which enforce Directive 2010/63/EU (Directive 2010/63/EU of the European Parliament and of the Council of 22 Sep. 2010 on the protection of animals used for scientific purposes). Accordingly, the project has been authorized by the Italian Competent Authority (Ministry of Health).
Wild-type (wt) mice (C57BL/6J) were purchased from the Charles River Laboratories. Terc+/− mice (B6. Cg-Terctm1 Rdp/J Stock No: 004132 | mTR−/−) (Blasco et al, 1997) were purchased from Jackson Laboratory. Terc+/− mice were intercrossed to generate first-generation (G1) homozygous Terc−/− knockout mice. Second-generation (G2) Terc−/− mice were generated by successive breeding of G1 Terc−/− and then G3 Terc−/− mice by crosses between G2 Terc−/− mice. Finally, wild-type C57BL/6J and G3 Tert−/− mice were used for the experiments. The 8- to 10-week-old mice were injected intraperitoneally (i.p.) with a control, anti-teloG, and anti-teloC ASO at a final concentration of 15 mg/kg twice a week for 4 weeks. The animals were sacrificed at 12 months of age. The lungs were collected, and part of it was snap-frozen for RNA extraction and part was washed in PBS and collected for fixation in 10% neutral-buffered formalin overnight, washed in water, and paraffin-embedded for histological analysis.
Antisense oligonucleotides (ASOs) sequences: The locked nucleic acid-modified oligonucleotides with a fully phosphorothioate backbone were produced by Qiagen as described (Rossiello et al, 2017).
Sequences were as follows (5′-3′ orientation):
Formalin-Fixed Paraffin-Embedded (FFPE) samples preparation: Lungs, femur and spleen samples were fixed in 10% Neutral Buffered Formalin (Diapath S.p.A., #F0043) at 4° C. overnight and then washed in running water at slow flux for 5h. Following fixation, the femurs were incubated in Microdec, EDTA-based-1000 ml (Diapath S.p.A., #D0053) for 2-3h for decalcification and then washed in running water at slow flux for 2 h. Then, all the samples were transferred to 70% ethanol, dehydrated, and embedded in paraffin.
Histopathological examination and airspaces analysis for the lungs: Paraffin-embedded tissues were cut into 4-μm sections and routinely stained with hematoxylin and eosin (H&E) for histopathological analysis. For the histopathological evaluation, a semiquantitative scoring system was utilized, which involved the following factors: inter-alveolar septal thickening, interstitial cellularity increase, interstitial inflammatory infiltration, intra-alveolar inflammatory infiltration and erythrocyte extravasation. All the factors were morphologically evaluated and scored according to the degree of severity (0, normal; 1, slight; 2, moderate; 3, marked) and extension (0, absent; 1, focal; 2, multifocal; 3, diffuse). An overall morphological and pathological score was calculated for each sample as the product of the degree of severity and the extension. To determine the number of airspaces in mouse lung samples, slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems) and a semiquantitative analysis was conducted on five nonoverlapping fields per sample, using a magnification of 20×.
Histopathological examination for bone marrow and spleen: Paraffin-embedded tissues were cut into 4-μm sections and routinely stained with hematoxylin and eosin (H&E) for histopathological analysis. For the histopathological evaluation of bone marrow phenotype, a semiquantitative scoring system was applied, which included the following variables: erythroid colonies contraction, myeloid cell expansion (segmented), myeloid cell expansion (immature), megakaryocyte clustering, megakaryocyte pleiomorphism and erythro/hemophagocytosis. For the histopathological evaluation of spleen phenotype, a semiquantitative scoring system was applied, which included the following variables: white pulp effacement, red pulp hyperplasia, myeloid/erythroid precursors increase, megakaryocyte hyperplasia and clustering and hemosiderin-laden macrophages. All the variables were morphologically evaluated and scored according to the degree of severity (0, normal; 1, slight; 2, moderate; 3, marked) and extension (0, absent; 1, focal; 2, multifocal; 3, diffuse). An overall morphological and pathological score was calculated for each sample as the product of the degree of severity and the extension.
Masson's Trichrome for the lungs: Masson's Trichrome staining was performed with Masson's trichrome Stain kit (DiaPath) following manufacturer's protocol. Masson Trichrome staining is employed to identify lung fibrosis. This staining method highlights collagen fibers in blue and muscular fibers in red. Slide digitalization was performed using an Aperio CS2 digital scanner (Leica Biosystems) with the ImageScope software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems).
To quantify the lung fibrosis, the “Area Quantification v2.4.2” algorithm of HALO software (Indica Labs) was utilized specifically for quantifying the blue-colored collagen fibers. This algorithm enabled the evaluation of Masson's trichrome staining in five separate fields, without overlap, using a medium-power magnification (×200) and independently of cell segmentation.
Immunolocalization and quantitative analyses for mouse paraffin embedded tissues: 4-μm FFPE tissue sections were deparaffinized, rehydrated, and unmasked using Novocastra Epitope Retrieval Solutions pH9 in thermostatic bath at 98° C. for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H2O2 and Fc blocking by a specific protein block (Leica Novocastra), the samples were incubated with the primary antibodies:
Immunohistochemistry (IHC) was developed using anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody HRP (Invitrogen, 1:500) and DAB (3,30-Diaminobenzidine, Novocastra) as substrate chromogen. Double IHC staining was performed by applying SignalStainBoost IHC Detection rabbit (cod. #18653, Cell Signaling Technology) alkaline phosphatase-conjugated and Vulcan Fast Red as substrate chromogen. Nuclei were counter-stained with Gill's hematoxylin No. 1 (BioOptica). For double-marker immunofluorescence (IF), in order to multiplex antibodies raised in the same species (p16 and proSP-C), Opal Multiplex IHC kit was developed. After deparaffinization, antigen retrieval was performed using microwave heating in pH9 buffer and the first primary antibody was incubated. Immunofluorescence labeling was achieved by incubating with a specific secondary antibody followed by the addition of Opal 520 fluorophore-conjugated tyramide signal amplification (TSA) and microwave treatment in pH9 buffer. The slides were again processed with the microwave treatment to strip primary/secondary antibody complex and allow the next antigen-antibody staining. Another round of staining was performed using Opal 620 fluorophore-conjugated tyramide signal amplification and DAPI nuclear counterstain. Slides were analyzed under a Zeiss Axioscope A1 microscope equipped with four fluorescence channels widefield IF. Microphotographs were collected using a Zeiss Axiocam 503 Color digital camera with the Zen 2.0 Software (Zeiss). Quantitative analyses of γH2AX, 53BP1 and CD45 IHC mouse stainings were performed by calculating the average percentage of positive signals in five nonoverlapping fields at medium-power magnification (×200) using the Nuclear v9 or Positive Pixel Count v9 ImageScope software. Double IHC (γH2AX and proSP-C; 53BP1 and proSP-C) and double IF (p16 and proSP-C) stainings were analyzed through the use of the segmentation-based algorithm “Multiplex IHC v3.2.3” or “HighPlex FL v4.2.3” of HALO software (Indica Labs), quantifying the average percentage of γH2AX+ or 53BP1+ or p16+ cells in type II pneumocytic pro-SP-C+ cells in five nonoverlapping fields at medium-power magnification (x200).
Antibodies for lung, spleen and bone marrow: The following primary antibodies were used for IHC and IF on mouse tissues: rabbit polyclonal anti-γH2AX (phospho S139) (1:1000 pH6, AB11174, Abcam), rabbit polyclonal anti-53BP1 (1:1000, pH6, NB100-304, Novus), rabbit polyclonal Anti-CD45 antibody (1:500, pH9, AB10558, Abcam), rabbit monoclonal anti-ARPC5/p16 [EP1551Y] (1:100, pH9, AB51243, Abcam), rabbit polyclonal anti-Prosurfactant Protein C (proSP-C) (1:200, pH9, AB3786, Millipore). Rabbit Recombinant Monoclonal CD41 antibody (Abcam, #ab181582; 1:250, pH9); Rabbit Recombinant Monoclonal Iba1 antibody (Abcam, #ab178847; 1:2000, pH9); Rabbit Polyclonal Myeloperoxidase antibody (Abcam, #ab9535; 1:25, pH9); Rabbit Recombinant Monoclonal PAX5 antibody (Abcam, #ab109443; 1:1000, pH9); Rat monoclonal anti-Ly76 (Ter119) antibody (Abcam, #ab91113; 1:50, pH9) or BD Pharmingen™ Purified Rat Anti-Mouse TER-119/Erythroid Cells (BD Biosciences, 553671; 1:50, pH9).
RNA extraction from mouse tissues: To isolate RNA, 20-30 mg lung tissue was homogenized in TRIzol (Life Technologies) with Tissue Lyzer II (Qiagen) and processed with RNeasy Kit (Qiagen) according to the manufacturer's specifications. To increase the purity of the RNA extracted, a convenient on-column DNase (Qiagen) treatment was performed to remove the residual amounts of DNA. NanoDrop spectrophotometer was used to detect RNA quantity and purity. RNA purity was ascertained via NanoDrop 260/280 and 260/230 ratios.
Illumina Stranded mRNA sequencing: The abundance and integrity of total RNA was assessed using Qubit fluorimeter an Agilent Bioanalyzer 2100 instrument (Agilent Technologies, Palo Alto, CA). For each sample, 500 ng of total RNA were used to generate a library of fragments using Illumina Stranded mRNA prep ligation kit. Oligo (dT) magnetic beads purify and capture the mRNA molecules containing polyA tails. The purified mRNA was fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In a second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. The final steps added adenine (A) and thymine (T) bases to fragment ends and ligate adapters. The resulting products were purified and selectively amplified with 12 cycles of PCR to generate an indexed library of fragments. The library was quantified using Qubit 4.0 fluorimeter, checked on Agilent Bioanalyzer 2100 then loaded on Illumina NextSeq550Dx sequencer for sequencing, following the manufacturer's instructions.
Library fragments were sequenced using 2×75 nt readmode, thus sequencing 75 nucleotides from both ends of each fragment; on average, ˜50 Million Paired-end fragments were sequenced for each sample. Sequencing results were generated in fastq.gz format.
RNA-sequencing analysis: Reads were aligned and count with the NF-core/rnaseq pipeline (v3.12.0-g3bec233). The reference genome on mm10 genome was used. The quality check was performed with FastQC and SampleNetwork. The differential analysis was performed on R with DESeq2, differentially expressed genes were defined as genes with p-value adjusted (Benjamini-Hochberg)<0.05. The Pathway Analysis was done with fsgea package. The hallmark pathways are from the Mouse MSigDB Collection. All the plots were done with ggplot, ComplexHeatmap and clusterprofiler packages.
Competitive BM transplant: The protocol of competitive BM transplant was adapted from a previously published protocol (Kwarteng and Heinonen J Vis Exp. 2016). Briefly, CD45.1+CD45.2+ recipient mice were bred in house by crossing CD45.2+ C57Bl/6 mice with congenic CD45.1+ B6.SJL mice and lethally irradiated with two doses of 4.5 Gy (the first dose was given the day before transplantation and the second 2-3 hours before transplantation). BM from CD45.1+ and CD45.2+ donor mice was harvested by flushing tibias and femurs, washed once in PBS and processed for flow cytometry staining to calculate the percentage of HSPCs as defined by the markers Lin−cKit+Sca1+, in order to inject the same quantities of HSPCs from CD45.1+ and CD45.2+ donors into the recipients. Subsequently, BM cells from CD45.1+ and CD45.2+ were mixed in a 1:1 ratio and injected intravenously into the lateral tail vein of the lethally irradiated recipients. Peripheral blood (100 ul) was collected from the lateral tail vein 4-, 8-, 12-weeks post-transplant and analyzed by flow cytometry using the following antibodies: Brilliant Violet 421™ anti-mouse CD45.1 Antibody (BioLegend, #110731) and PerCP anti-mouse CD45.2 Antibody (BioLegend, #109825). Analysis was performed using a Cytek Aurora 5 spectral flow cytometer together with FlowJo (TreeStar Inc) software.
Flow Cytometry Analysis: BM was harvested from mice by flushing tibias and femurs. Spleen was harvested from mice and smashed in PBS. Single-cell suspensions from both organs were washed once in PBS and RBCs were eliminated using RBC Lysis Buffer (BioLegend, #420301). LSK cells and the immune subpopulations were analyzed by immunophenotyping using the following antibodies: Pacific Blue™ anti-mouse Lineage Cocktail (BioLegend, #133310); PE anti-mouse CD117 (c-kit) Recombinant Antibody (BioLegend, #155106); APC anti-mouse Ly-6A/E (Sca-1) Antibody (BioLegend, #108112); Brilliant Violet 605™ anti-mouse/human CD45R/B220 Antibody (BioLegend, #103244); Pacific Blue™ anti-mouse CD19 Antibody (BioLegend, #115526); Brilliant Violet 510™ anti-mouse IgD Antibody (BioLegend, #405723); APC/Fire™ 750 anti-mouse IgM Antibody (BioLegend, #406538); APC anti-mouse/human CD11b Antibody (BioLegend, #101212); Brilliant Violet 570™ anti-mouse Ly-6C Antibody (BioLegend, #101212); Brilliant Violet 785™ anti-mouse Ly-6G Antibody (BioLegend, #127645).
Intracellular protein levels were assessed after cell permeabilization with True-Nuclear™ Transcription Factor Buffer Set (BioLegend, #424401) following manufacturer instruction and using the following antibodies: Histone H2AX [p Ser139] Antibody [PerCP] (NovusBio, #NB100-384PCP); KAP1 [p Ser824] Antibody [Alexa Fluor®488] (NovusBio, #NBP2-32073AF488). Analysis was performed using a Attune NxT or Cytek Aurora 5 spectral flow cytometer together with FlowJo (TreeStar Inc) software
Colony Forming Unit Assay: BM cells (1×105) isolated from mice were cultured in methylcellulose-based medium (Stem Cell Technologies, MethoCult M3434) and scored for colonies after 7 days.
Cells and treatment: Mouse hippocampal primary neurons established from postnatal day 0-2 pups were plated on dishes or glass coverslips coated with poly-L-lysine (Sigma-Aldrich). Cultures were used for experiments after 14 days in vitro (DIV) and incubated at 37° C. during all treatments in neurobasal medium supplemented with B27, Glutamax and Penicillin/Streptomycin.
Human cortical neurons derived from IPCs. Human iPSCs were purchased from HIPSCI(HPSI0613i_nukw_1) and cultured as colonies on Matrigel hESC-qualified (Corning)-coated plates in mTESR-plus (VODEN). For the differentiation the Studer's protocol was applied as described in Qi et al (Yuchen Qi et al).
For both mouse and human neurons, anti-Telo G and anti-Telo C (10 μM) ASOs were delivered naked to the cell medium for 24 hours, followed by the addition of Aβ oligomers (10 μM) or a vehicle. After 48 hours from Aβ oligomers administration, the cells were processed for immunofluorescence, RNA extraction, and viability assays
Aβ42 oligomers preparation: AB1-42 (Aβ42 oligomers) oligomers were obtained from a depsi-peptide AB1-42 which was purchased from GenScript. At variance with the native peptide, the depsi-peptide is highly soluble and it has a much lower propensity to aggregate, thus preventing the spontaneous formation of seeds in the solution (Beeg et al). The native Aβ1-42 peptide was then obtained from the depsi-peptide by a “switching” procedure in basic conditions. The alkaline stock solution (400 μM) was diluted to obtain 100 μM Aβ1-42 oligomers in phosphate buffer and incubated for 18-20 h at 4° C. Aβ1-42 preparations were diluted in the medium to reach the final concentration (10 μM). Before adding oligomers to the medium Hepes IM was add to the medium to reach the final concentration (80 μM).
To confirm the state of the Aβ1-42 oligomers, we performed Western blot analysis. Briefly, the oligomers solution was subjected to 4-12% Tris-Tricine Gel (Invitrogen) electrophoresis and transferred to polyvinyldene difluoride (PVDF) membranes (Millipore, Billerica, MA). These membranes were incubated with a primary antibody against mouse monoclonal human Aβ (6E10) and anti-oligomers A11. For detection, the membrane was incubated with a horseradish-peroxi-dase-conjugated Ig anti-mouse antibody. Immunoreaction signals were visualized with SuperSignal™ Pierce™ West Pico PLUS (Thermofisher).
Immunofluorescence on neurons: Cells were fixed with 4% PFA. After incubation with blocking solution, cells were stained with primary antibody for O.N. at 4° RT, washed and incubated with secondary antibodies for 45 min at RT. Nuclei were stained with 4,6-diamidino-2-pheny-lindole (DAPI; 1 mg ml-1). Samples were mounted in mowiol. Image acquisition was performed in a Leica SP5 confocal microscope. The detection parameters were set in the control samples and were kept constant across specimens.
ImmunoFISH on neurons: After the immunofluorescence protocol, mouse primary neurons were treated as follows. After incubation with the secondary antibodies, cells were washed, fixed with PFA 4%+triton 0.1% for 10 min RT, incubated with glycine 10 mM for 30 min RT, washed, denatured at 80° C. for 5 min and incubated with Cy5-conjugated TeloC PNA probe (F1003-Panagene). After washes, nuclei were stained with 4,6-diamidino-2-pheny-lindole (DAPI; 1 mg ml-1). Samples were mounted in mowiol. Image acquisition was performed in a Leica SP5 confocal microscope. The detection parameters were set in the control samples and were kept constant across specimens.
Antibodies for mouse primary neurons and human cortical neurons: The following primary antibodies were adopted for IF on mouse and human cells: MAP2 (Chicken Polyclonal MAP2 antibody ab92434, Abcam); gH2AX (Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718, Cell Signaling; Anti-phospho-histone H2A.X, ser139 Mouse 05-636. Millipore), 53BP1 (53BP1 (H-300), Rabbit, Sc-22760 Rabbit; 53BP1 Antibody, rabbit A300-272A, Bhetyl); pATM (Anti-phospho-ATM (Ser1981) Antibody, clone 10H11.E12, mouse #05-740, Millipore); pSP/Q (Anti-phospho-(Ser/Thr) ATM/ATR substrate antibody, rabbit #2851L, Cell signaling).
RNA extraction from mouse primary neurons and human cortical neurons: total RNA was extracted using Maxwell® RSC Instrument, with Maxwell® RSC simplyRNA Tissue Kit (AS134, Promega), following manufacturer's instructions. NanoVue™ Plus Spectrophotometer (GE Healthcare) was used to detect RNA quantity and purity. RNA purity was ascertained via Nano Vue 260/280 and 260/230 ratios.
Reverse Transcription Quantitative PCR (RT-qPCR) mouse primary neurons and human cortical neurons: 1-2 μg of total cell RNA was reverse transcribed into cDNA using SuperScript VILO cDNA Synthesis Kit (11754050, ThermoFisher). A volume corresponding to 25-50 ng of cDNA was used for each RT-qPCR reaction using Roche LightCycler 480 SYBR Green I Master (04707516001, Roche) sequence detection system. Each reaction was performed in triplicate.
Primers Sequences (5′-3′ Orientation) were:
Strand-specific real-time quantitative PCR Detection: Detection of tdilncRNAs was performed as previously described (Rossiello et al, 2017), with some modifications. Briefly, RNA samples were treated with DNase I (Thermo Scientific) at 37° C. for 1 h. Next, 1 μg of total RNA was reverse transcribed using the Superscript First Strand cDNA synthesis kit (Invitrogen) with strand-specific primers. cDNA was passed on a MicroSpin™ G-50 columns (Cytiva) and qPCR was performed using SYBR Green I Master Mix (Roche). A volume of cDNA corresponding to 20 ng of initial RNA was used. Each reaction was performed in triplicate. Rplp0 was used as a control gene for normalization.
qPCR Primer Sequences (5′-3′ Orientation):
Cell Viability: Neuronal viability was evaluated by ATP detection (Cat #G9242, CellTiter-Glo.2 Luminescent Cell Viability Assay; Promega, Madison, WI, USA) accordingly to the manufacturer instructions. Briefly cells were plated on 96-wells plate and were lysed and incubated with CellTiter-Glo® 2.0 Reagent for 10′ and luminescence were recorder with EnVision multilabel plate reader.
Statistical analyses: Results are shown as mean±standard error of the mean (SEM). P-values were calculated by the Prism software.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15178809.8 | Jul 2015 | EP | regional |
This application is a continuation-in-part of U.S. patent application Ser. No. 17/065,409, filed on Oct. 7, 2020, which is a continuation of U.S. patent application Ser. No. 15/748,133, filed on Jan. 26, 2018, which is the U.S. National Phase under 35 U.S.C. § 171 of International Patent Application PCT/EP2016/068162, filed Jul. 29, 2016, which claims priority to European Patent Application No. EP 15178809.8, filed Jul. 29, 2015, the entire content of each of which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15748133 | Jan 2018 | US |
| Child | 17065409 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17065409 | Oct 2020 | US |
| Child | 18888819 | US |
