Patent Application
20250176509
This invention falls within the field of molecular biology, biotechnology and medicine. More particularly, it relates to non-human animals and to compositions and methods useful for the treatment of conditions associated with short telomere length. More particularly, it relates to compositions and methods useful for the treatment of conditions associated with kidney fibrosis associated to short telomere length.
Telomeres are specialized structures at the ends of chromosomes, which have a role in protecting the chromosome ends from DNA repair and degrading activities. Mammalian telomeres consist of TTAGGG repeats bound by a multi-protein complex known as shelterin. A minimum length of TTAGGG repeats and the integrity of the shelterin complex are necessary for telomere protection, as shortened telomeres have been associated with numerous diseases. These structures are essential for chromosome integrity by preventing telomere fusions and telomere fragility. Telomere length is controlled by the ribonucleoprotein enzyme telomerase which can de novo add telomeric sequences onto telomeres. Telomerase is a cellular reverse transcriptase (TERT, telomerase reverse transcriptase; also known as TP2; TRT; EST2; TCS1; hEST2) capable of compensating telomere attrition through de novo addition of TTAGGG repeats onto the chromosome ends by using an associated R A component as template (Terc, telomerase RNA component). Telomerase is expressed in most adult stem cell compartments; however, this is not sufficient to maintain telomere length as evidenced by the fact that telomere shortening occurs with age in most human and mouse tissues.
Because telomeric sequence is naturally lost upon every cell division (known as the end replication problem) and somatic cells express telomerase at very low levels or not at all telomeres shorten throughout life. When telomeres become critically short, they lose their protective function and a persistent DNA damage response at the telomeres is triggered which subsequently leads to a cellular senescence response (Harley et al., 1990, Flores et al., 2008).
Accumulation of short telomeres is a hallmark of aging. Mutations in telomerase or telomere-binding proteins lead to telomere shortening or dysfunction and are at the origin of human pathologies known as ‘telomere syndromes’, which are characterized by loss of the regenerative capacity of tissues and fibrotic pathologies and which include some cases of aplastic anemia, dyskeratosis congenita and fibrosis of the lung. Adeno-associated viruses (AAV) based telomerase gene therapy was found to be beneficial to extend health span, in the context of normal physiological aging in wild-type mice. Adult and aged mice were subjected to AAV9-mTERT gene therapy to broadly express the catalytic subunit of mouse telomerase (mTERT). The health span of the TERT treated mice was significantly increased, and aging was decelerated, as indicated by a number of physiological parameters (glucose and insulin tolerance, osteoporosis, neuromuscular coordination, rota-rod, etc). In addition, their mean lifespan, compared to control groups, was increased by 24% and 13% in adult an old mice, respectively. A single intravenous administration of AAV9-TERT in adult mice resulted in an increase in telomere length in peripheral blood cells (Bernardes de Jesus et al., 2012). These results highlight the importance of mouse models for the study of the specific pathologies and the potential therapeutic approaches.
Chronic kidney disease (CKD) is a disorder with high mortality, and its incidence is increasing owing to the demographic aging phenomenon. Kidney fibrosis is the primary determinant of end-stage renal disease characterized by fibroblast activation and excessive production and deposition of extracellular matrix (ECM), leading to destruction of renal parenchyma, an inflammatory and fibrotic response and decreased renal function. It is believed that short telomeres can contribute to kidney fibrosis. However, the role of short telomeres in kidney fibrosis remains less understood, in part due to the lack of appropriate mouse models. Further, it is not known whether telomerase-deficient mice with short telomeres will develop kidney fibrosis or they require additional insults to contribute to the disease. In the past, we have developed mouse models that develop aplastic anemia, and lung fibrosis associated with short telomeres. These mouse models demonstrate the role of short telomeres in the origin of these diseases and point to potential therapeutic strategies.
In the present invention, we develop suitable mouse model to study kidney fibrosis associated with short telomeres, which allows us to investigate whether telomerase activation can be an effective treatment to attenuate kidney fibrosis associated with short telomeres.
The invention provides compositions and methods useful for the treatment and prevention of kidney fibrosis associated with short telomeres.
In one aspect, the present invention provides a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT) for use in treating kidney fibrosis associated to the presence of short telomeres. Preferably, TERT is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, TERT is encoded by a nucleic acid sequence consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, TERT comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, TERT consists of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives the expression of the coding sequence. Preferably, the vector is a non-integrative vector. Preferably, the vector is a ribonucleic acid (RNA), preferably a messenger RNA.
Preferably, wherein the vector is an adeno-associated virus-based non-integrative vector. Preferably, the vector is an adeno-associated virus-based vector derived from a serotype 9 adeno-associated virus (AAV9). Preferably, the capsid of the adeno-associated virus-based vector is made of capsid proteins of the serotype 9 adeno-associated virus (AAV9), and the nucleic acid sequence contained in the capsid is flanked at both ends by internal terminal repeats corresponding to serotype 2 adeno associated viruses. Preferably, the nucleic acid contained in the capsid comprises a fragment which encodes the amino acid sequence coding for TERT. Preferably, the vector comprises a regulatory sequence which is a constitutive promoter, preferably the cytomegalovirus (CMV) promoter.
In a further aspect, the present invention provides a A non-human animal characterized in that it exhibits a pathological condition of kidney fibrosis, wherein the non-human animal is obtained or obtainable when a sublethal dose of folic acid is administered to a non-human animal whose germ cells comprise a hereditary deactivation of both alleles of the Tert gene. Preferably, the animal is a mammal, preferably a rodent. The sublethal dose of folic acid is preferably a dose of up to 200 mg/kg body weight, preferably of 125 mg/kg body weight.
Preferably, the folic acid is administered intraperitoneally. Preferably, the folic acid is administered when the animals are between 4-10 weeks of age, preferably between 6-8 weeks of age. Preferably, wherein the folic acid is administered once.
Preferably, the non-human animal whose germ cells comprise a hereditary deactivation of both alleles of the Tert gene (Tert−/−) is the third generation (G3) of Tert−/− lineage.
The role of short telomeres in aplastic anemia and lung fibrosis was found to be important mainly due to the development of appropriate mouse models, which opened the path for the development of potential therapeutic strategies against said diseases. However, other complex diseases, such as kidney fibrosis, lack of suitable animal models, hampering the study of the role of short telomeres in said pathologies.
In the present study, we aimed at studying the involvement of short telomeres in kidney fibrosis. In order to do that, suitable animals models had to be developed. We first analysed the kidneys of wild-type mice, as well as mice lacking the telomerase catalytic subunit, Tert, that were bred for three generations (G3) and thus are G3 Tert−/− mice (
Folic acid (FA) induces interstitial fibrosis, but only at high doses. High dosages of folic acid (250 ug/g BW) given i.p in mice induces folic acid crystals rapidly with tubular necrosis in the acute phase (1-14 days) and patchy interstitial fibrosis in the chronic phase (28-42 days). However, lower doses of FA were not found to induce kidney fibrosis in wild-type mice. In view of this, we aimed to study the effect of a sublethal dose of FA in Tert+/+ and G3 Tert−/− mice aged 8-9 weeks, and its contribution to the development of kidney fibrosis associated to short telomeres. To this end, we first subjected Tert+/+ and G3 Tert−/− mice aged 8-9 weeks to increasing doses of FA (50, 100, 125 and 250 mg kg-1 body weight;
In addition, to assess the contribution of dysfunctional telomeres to the induction of kidney fibrosis, we used a second model of telomere dysfunction by deleting TRF1, one of the components of the shelterin telomere protective complex. In particular, we used a mouse model in which treatment with tamoxifen led to deletion of Trf1 in all kidney cells (
Overall, the present invention provides suitable mice models suitable to study kidney fibrosis associated to the telomere length. Said mouse models are key tools to understand the role of short and dysfunctional telomeres and resulting DNA damage in the molecular events associated with fibrosis, particularly kidney fibrosis. They will also allow the development of gene therapy approaches aimed at correcting the accumulation of critically short telomeres that is associated with said conditions.
In view of the above, the present invention provides, in a first aspect, compositions and methods useful for the treatment and prevention of a condition associated with short telomere length, particularly kidney fibrosis, to a subject in need thereof. A “condition associated with short telomere length” is one which is characterized by an accumulation of critically short telomeres. In certain embodiments, the condition associated with short telomere length is characterized by mutations in a gene or genes involved in telomere maintenance. Specific examples of such genetically based conditions include, but are not limited to, kidney fibrosis, preferably kidney fibrosis associated with telomere shortening. Short telomeres exacerbate epithelial-to-mesenchymal transition (EMT) program in the kidneys and thus promote pathological scarring of kidney tissue, i.e. fibrosis. Kidney fibrosis associated with telomere shortening is characterised by the presence of short/dysfunctional telomeres owing to mutations in genes related to telomere maintenance, being the most frequently mutated those encoding proteins of the telomerase complex (i.e. TERT, TERC, NOP10, DKC1, NHP2). Both a functional telomerase complex and a proper telomere capping structure by the shelterin proteins are required for maintenance and capping of chromosome ends, respectively.
In some embodiments, the kidney fibrosis is characterised by abnormal production and deposition of extracellular matrix (ECM) proteins mainly in the kidney interstitium and results in structural damage, impairment of renal function, and eventually end-stage renal disease (ESRD). The clinical features of patients suffering from kidney fibrosis are swollen ankles, feet or hands, shortness of breath, tiredness, blood in urine, insomnia, itchy skin, muscle cramps, and headache, among others. Kidney fibrosis is a direct consequence of the kidney's limited capacity to regenerate after injury. In some embodiments, kidney fibrosis is characterized by fibroblast activation and excessive production and deposition of extracellular matrix (ECM), leading to destruction of renal parenchyma, an inflammatory and fibrotic response and decreased renal function. Please note that kidney fibrosis is synonymous of renal fibrosis.
Accordingly, the invention provides compositions and methods for treating a subject in need thereof who is suffering from a condition associated with short telomere length, preferably kidney fibrosis, comprising administering to the patient an agent which increases the telomere length of the patient. The present invention also relates to method of preventing said condition. “Preventing”, “to prevent” or “prevention” or any other similar term, include without limitation, decreasing, reducing or ameliorating the risk of a symptom, disorder, condition, or disease, and protecting an animal from a symptom, disorder, condition, or disease. A prevention may be applied or administered prophylactically. “Treating”, “to treat” or “treatment” or any other similar term, include without limitation, restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, clinical sign, disorder, condition, or disease. A treatment may be applied or administered therapeutically.
“Subjects in need thereof” refers herein to subjects suffering from a condition that exhibit premature onset of pathologies resulting from a defective regenerative capacity of kidney tissues. In certain embodiments, subjects in need of said treatment exhibit a fibrotic response, preferably kidney fibrotic response, and/or a decreased renal function. In some embodiments, subjects in need of said treatment present a disease or condition that implies or eventually leads to kidney fibrosis, such as glomerulosclerosis, renal interstitial fibrosis, diabetes, hypertension, infectious glomerulonephritis, renal vasculitis, ureteral obstruction, genetic alterations, autoimmune diseases, or any combination thereof. Preferably, the subject in need of said treatment suffers from kidney fibrosis, preferably kidney fibrosis associated with telomere shortening. The terms “patient” or “subject” are considered herein synonymous and refer to a mammal. In certain embodiments the patient is a rodent, primate, human, ungulate, cat, dog, mouse, rat, rabbit, pig, horse, sheep, cow, domestic cat or dog, or other domestic pet or domesticated mammal. In a preferred embodiment, the patient or the subject in need thereof is a human.
In one embodiment, the agent prevents degradation of the chromosomal ends. In one embodiment, the agent increases the activity of telomerase reverse transcriptase (TERT). In one embodiment, the method of treatment is a gene therapy method that comprises administering to the patient a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT).
In certain embodiments, the TERT sequence used in the gene therapy vector is derived from the same species as the subject. For example, gene therapy in humans would be carried out using the human TERT sequence. In one embodiment, the TERT is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 (human TERT variants 1 and 2), or is an active fragment or functional equivalent of SEQ ID NO: 1 or SEQ ID NO: 3. The polypeptide sequence encoded by SEQ ID NO: 1 is set forth in SEQ ID NO: 2. The polypeptide encoded by SEQ ID NO: 3 is set forth in SEQ ID NO: 4. As used herein, “functional equivalent” refers to a nucleic acid molecule that encodes a polypeptide that has TERT activity or a polypeptide that has TERT activity. The functional equivalent may displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared to TERT encoded by SEQ ID NO: 1 or SEQ ID NO: 3. In an embodiment, the TERT is encoded by a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Functional equivalents may be artificial or naturally-occurring. For example, naturally-occurring variants of the TERT sequence in a population fall within the scope of functional equivalent. TERT sequences derived from other species also fall within the scope of the term “functional equivalent”, in particular the murine TERT sequence given in SEQ ID NO: 5. In a particular embodiment, the functional equivalent is a nucleic acid with a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3. In a further embodiment, the functional equivalent is a polypeptide with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to SEQ ID NO: 2 or SEQ ID NO: 4. In an embodiment, the TERT comprises or consists of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In the case of functional equivalents, sequence identity should be calculated along the entire length of the nucleic acid. Functional equivalents may contain one or more, e.g. 2, 3, 4, 5, 10, 15, 20, 30 or more, nucleotide insertions, deletions and/or substitutions when compared to SEQ ID NO: 1 or SEQ ID NO: 3. The term “functional equivalent” also encompasses nucleic acid sequences that encode a TERT polypeptide with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity to the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4, but that show little homology to the nucleic acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 3 because of the degeneracy of the genetic code. Sequence identity may be calculated by any one of the various methods in the art, including for example BLAST and variations on these alignment programs.
As used herein, the term “active fragment” refers to a nucleic acid molecule that encodes a polypeptide that has TERT activity or polypeptide that has TERT activity, but which is a fragment of the nucleic acid as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4. An active fragment may be of any size provided that TERT activity is retained. A fragment will have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% identity to SEQ ID NO: 1-4 along the length of the alignment between the shorter fragment and SEQ ID NO: 1-4.
Fusion proteins including these fragments can be comprised in the nucleic acid vectors needed to carry out the invention. For example, an additional 5, 10, 20, 30, 40, 50 or even 100 amino acid residues from the polypeptide sequence, or from a homologous sequence, may be included at either or both the C terminal and/or N terminus without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit biological activity.
In one embodiment, the method of treatment is a gene therapy method and/or the nucleic acid vector used is a gene therapy vector. Gene therapy methods and vectors are well known in the art and generally comprise delivering a nucleic acid encoding a therapeutically active protein to a subject. The nucleic acid may be delivered in a number of ways and forms including delivering naked deoxyribonucleic acid (DNA) such as plasmid or mini-circles, delivering ribonucleic acids (RNA), such as messenger RNA, the use of liposomes or cationic polymers or other engineered nano-particles containing the nucleic acid, or viral vectors that encapsidate the nucleic acid.
In a further embodiment, the gene therapy is achieved using stable transformation of organisms with an inducible expression system. Suitable inducible expression systems are known in the art and include the CRE-LOX recombinase based system which is suitable for use in mice and tetracycline-regulated which can be used in the treatment of human subjects.
In one embodiment the gene therapy vector is a ribonucleic acid (RNA) vector comprising or consisting of the coding sequence for telomerase reverse transcriptase (TERT), as defined above. In some embodiments, the RNA nucleic acid vector is delivered in a naked form, i.e., unshielded RNA. In some other embodiments, the RNA nucleic acid vector is modified as to be protected from nuclease degradation. Said modifications include chemical modifications in the structure of the RNA, such as those performed in the 2′ position, in the phosphate linkage and/or in the nucleobase. Other modifications include the encapsidation of the RNA nucleic acid vector into nanoparticles, polymers, endosomes, lipids and lipid-like particles, or other delivery vectors, such as viruses. Nanoparticle encapsulation of RNA physically protects nucleic acids from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape. Polymers include Poly(β-amino esters), poly-L-lysine, polyamidoamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to RNA delivery. Chemically well-defined means of delivery is to directly conjugate a bioactive ligand, such as N-acetylgalactosamine, cholesterol, vitamin E, antibodies, peptides, to the RNA that will allow it to enter the cell of interest.
Preferably, the RNA nucleic acid vector is a single stranded RNA molecule, more preferably a messenger RNA (mRNA) vector. The delivery of therapeutics mRNA vectors has been facilitated by maximizing the translation and stability of the mRNA vector, preventing its immune-stimulatory activity and the development of in vivo delivery technologies. In some embodiments, the mRNA vector is modified to increase the in vivo delivery efficacy. In an embodiment, the mRNA vector is modified to include the incorporation of a 5′ cap and/or a 3′ poly(A) tail to efficient translation and prolonged half-life of mature mRNAs. Cap analogues such as ARCA (anti-reverse cap analogues) and poly(A) tails of 120-150 bp can also be used. New types of cap analogues, such as 1,2-dithiodiphosphate-modified caps, with resistance against RNA decapping complex, are also included. The so-called codon optimization also facilitates better efficacy of protein synthesis and limits mRNA destabilization by rare codons. Similarly, engineering 3′ and 5′ untranslated regions (UTRs), which contain sequences responsible for recruiting RNA-binding proteins (RBPs) can enhance the level of protein product. N1-methyl-pseudouridine base modification is usually used to mask mRNA immune-stimulatory activity.
In one embodiment, the regulatory sequence operatively linked to the TERT coding sequence is the cytomegalovirus promoter (CMV), although other suitable regulatory sequences will be known to those of skill in the art. In other embodiments, the regulatory sequence operatively linked to the TERT coding sequence is a kidney-specific promoter. In a preferred embodiment, the coding sequence for telomerase reverse transcriptase (TERT) gene comprised in the RNA nucleic acid vector is operably linked to a CMV or a kidney-specific promoter, wherein the RNA nucleic acid vector further comprises a poly(A) sequence placed at the end of the coding sequence for the telomerase reverse transcriptase (TERT) gene.
In one embodiment the gene therapy vector is a viral vector. Viral gene therapy vectors are well known in the art. Vectors include integrative and non-integrative vectors such as those based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), Antiviruses, pox viruses, alphaviruses, and herpes viruses. Using non-integrative viral vectors, such as AAV, seems to be particularly advantageous. In one aspect, this is because non-integrative vectors do not cause any permanent genetic modification. Second, the vectors target to adult tissues, avoiding having the subjects under the effect of constitutive telomerase expression from early stages of development. Additionally, non-integrative vectors effectively incorporate a safety mechanism to avoid over-proliferation of TERT expressing cells. Cells will lose the vector (and, as a consequence, the telomerase expression) if they start proliferating quickly.
Particular examples of suitable non-integrative vectors include those based on adenoviruses (AdV) in particular gutless adenoviruses, adeno-associated viruses (AAV), integrase deficient lentiviruses, poxviruses, alphaviruses, and herpesviruses. Preferably, the non-integrative vector used in the invention is an adeno-associated virus-based non-integrative vector, similar to natural adeno-associated virus particles.
Vectors derived from adeno-associated viruses (AAVs) have emerged as one of the vectors of choice for many gene transfer applications because of their many desirable properties, including capability to transduce a broad range of tissues at high efficiency, poor immunogenicity and an excellent safety profile, toxicity being absent in many preclinical models. AAV vectors transduce post-mitotic cells and can sustain long-term gene expression (up to several years) both in small and large animal models of disease. Safety and efficacy of AAV gene transfer has been extensively studied in humans with encouraging results in the liver, muscle, CNS, and retina.
AAV preferentially target post-mitotic tissues, which are considered more resistant to cancer than the highly proliferative ones. Examples of adeno-associated virus-based non-integrative vectors include vectors based on any AAV serotype, i.e. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV. Tissue specificity is determined by the capsid serotype. Pseudotyping of AAV vectors and capsid engineering to alter their tropism range will likely be important to their use in therapy.
AAV2 is the best characterized serotype for gene transfer studies both in humans and experimental models. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes. AAV2 is therefore a good choice of vector to target these tissues, in particular when using the methods or vectors of the invention to treat a condition associated with one of these tissues. For example, treatment of neuromuscular degeneration may be targeted to skeletal muscle and/or neurons in this way.
Newly isolated serotypes, such as AAV7, AAV8, and AAV9 have been successfully adopted in preclinical studies, long term expression of the therapeutic gene is possible depending on the target tissue and the route of administration. In addition, the use of non-human serotypes, like AAV8 and AAV9, might be useful to overcome these immunological responses in subjects, and clinical trials have just commenced (ClinicalTrials.gov Identifier: NCT00979238).
Altogether, these encouraging data suggest that AAV vectors are useful tools to treat human diseases with a high safety and efficient profile.
The choice of adeno-associated viruses of wide tropism, such as those derived from serotype 9 adeno-associated virus (AAV9) is particularly advantageous when treating conditions associated with short telomere length. AAV9 viruses have shown efficient transduction in a broad range of tissues, with high tropism for liver, heart and skeletal muscle and thus the beneficial effects of gene therapy can be achieved in more tissues. In addition, AAV9 vectors have the unique ability to cross the blood-brain-barrier and target the brain upon intravenous injection in adult mice and cats (Foust et al Nature biotechnology 2009).
One aspect of the invention provides a system in which the capsid (which is the part of the virus which determines the virus tropism) of the adeno-associated virus-based vector is made of capsid proteins of the serotype 9 adeno-associated virus (AAV9). In one embodiment of the viral vectors for use in the invention, the polynucleotide sequence packed in the capsid is flanked by internal terminal repeats (ITRs) of an adeno-associated virus, preferably of serotype 2 which has been extensively characterised in the art and presents a coding sequence located between the ITRs. As set out above, the nucleic acid preferably codes for a functional TERT polypeptide. In one embodiment, the regulatory sequence operatively linked to the TERT coding sequence is the cytomegalovirus promoter (CMV), although other suitable regulatory sequences will be known to those of skill in the art. In another embodiment, the regulatory sequence operatively linked to the TERT coding sequence is a kidney-specific promoter.
When treating conditions associated with short telomere length, it is advantageous to target the treatment to the effected tissues. The choice of AAV serotype for the capsid protein of the gene therapy vector may be thus based on the desired site of gene therapy. If the target tissue is skeletal muscle, for example, in treating loss of neuromuscular coordination, AAV1- and AAV6-based viral vectors can be used. Both of these serotypes are more efficient at transfecting muscle than other AAV serotypes.
Alternatively, other viral vectors can be used in the present invention. Any vector compatible with use in gene therapy can be used in the present invention. Heilbronn & Weger (2010) Handb Exp Pharmacol. 197: 143-70 provides a review of viral vectors that are useful in gene therapy.
In accordance with all the previous discussion, vectors comprising a coding sequence for telomerase reverse transcriptase (TERT) suitable for use in gene therapy are an important point for putting the invention into practice. Suitable gene therapy vectors include any kind of particle that comprises a polynucleotide fragment encoding the telomerase reverse transcriptase (TERT) protein, operably linked to a regulatory element such as a promoter, which allows the expression of a functional TERT protein demonstrating telomerase reverse transcriptase activity in the targeted cells. Preferably, TERT is encoded by the nucleic acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or is an active fragment or functional equivalent of TERT. The term gene therapy vector includes within its scope naked DNA molecules such as plasmids or mini-circles, i.e. circular DNA molecules which do not contain bacterial DNA sequences, provided that the TERT coding sequence and its linked regulatory element are inserted in the plasmid, as well as to more complicated systems, such as particles with the structure of virions (viral particles), comprising at least a capsid and at least a polynucleotide sequence, with a size that allows the polynucleotide sequence to be packed within the capsid in a manner similar to that of the native genome of the virus of origin of the capsid. The polynucleotide sequence must include a region where the TERT coding sequence and its linked regulatory element are inserted such that the telomerase reverse transcriptase protein can be expressed from that polynucleotide sequence once the viral particle has infected a cell.
In one embodiment, the gene therapy vector suitable for being used in the invention is a non-integrative vector, such as an adeno-associated virus-based non-integrative vector. For the purposes of the invention, the choice of non-integrative vectors seems to be particularly advantageous, because they do not cause any permanent genetic modification. Also, as stated before, such vectors incorporate a safety mechanism to avoid over-proliferation of TERT expressing cells that will lose the vector if the cells start proliferating quickly.
Adeno-associated virus-based vectors derived from a serotype 9 adeno-associated virus (AAV9) are preferred because the beneficial effects can be achieved in more tissues (see above). In one particularly preferred embodiment, the regulatory sequence operatively linked to the TERT coding sequence is the cytomegalovirus promoter (CMV). The nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives the expression of the coding sequence. As used herein, the term “regulatory element” means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a nucleic acid sequence operably linked to the promoter. Such “regulatory elements” or “promoters” can control the expression of linked nucleic acid sequences either constitutively or inducible. The regulatory sequence may be a constitutive promoter. An example of a regulatory sequence which is a constitutive promoter is the cytomegalovirus (CMV) promoter.
The expression of TERT following gene therapy according to the invention persists for a time of several months to several years. In one embodiment of the invention, the subject is treated once. In an alternative embodiment, the subject is treated initially, and is then treated again once TERT expression levels decrease by about 50% of those attained immediately following treatment. Treatment may be repeated with the same or alternative vector to maintain the reduction in age-related disorders if necessary, for example annually, or once every 5 years or once a decade. When administering a second or subsequent dose, it may be necessary to use a different gene therapy vector, for example when using an AAV-based vector the second and subsequent administrations may be a vector with a capsid derived from a different serotype than that used for the first administration.
The methods of treatment of the invention have the effect of treating and/or preventing conditions associated with short telomere length. In a further aspect, therefore, the invention refers to a gene therapy method or the use of a nucleic acid vector as described above, for use in the treatment or prevention of kidney fibrosis, preferably kidney or fibrosis associated with short telomere length, in a subject in need thereof.
The effectiveness of treatment of the conditions associated with short telomere length can be measured by various methods known in the art. In one embodiment, the effectiveness of the treatment is measured by an increase in lifespan of a treated patient suffering from a condition associated with short telomere length as compared to the expected lifespan of an untreated patient suffering from the same condition. In certain embodiments, the lifespan is extended by 5%, 10%, 15%, 20% or more, with reference to the expected lifespan for a patient suffering from the same condition. In one embodiment, the effectiveness of the treatment is measured by a delayed or prevented kidney failure in a treated patient suffering from a condition associated with short telomere as compared to the expected onset kidney failure in an untreated patient suffering from the same condition. In certain embodiments, the delay in the onset of bone marrow failure of a treated patient suffering from a condition associated with short telomere length is extended by 5%, 10%, 15%, 20% or more, with reference to the expected onset of kidney failure for an untreated patient suffering from the same condition.
In one embodiment, the effectiveness of the treatment is measured by an increase in overall fitness of a treated patient suffering from a condition associated with short telomere length treated, preferably kidney fibrosis, as compared to the overall fitness of an untreated patient suffering from the same condition. Overall fitness can be determined by measuring physical attributes associated with the particular condition. Thus, an increase in overall fitness can be determined by a decrease in physical attributes associated with the particular condition exhibited by the treated patient. In one embodiment, increased overall fitness is measured by determining the estimated glomerular filtration rate (eGFR) and/or albuminuria (ACR). A glomerular filtration rate (GFR) of 60 or higher is in the normal range. A GFR below 60 may mean kidney disease. A GFR of 15 or lower may mean kidney failure. The standardized albumin-to-creatinine ratio (ACR) test shows whether you have albumin in your urine. A normal amount of albumin in your urine is less than 30 mg/g. Anything above 30 mg/g may mean you have kidney disease, even if your GFR number is above 60. In certain embodiments, the glomerular filtration rate in a treated patient is increased by 5%, 10%, 15%, 20% or more, with reference to the glomerular filtration rate of an untreated patient suffering from the same condition.
The efficacy of the treatment can also be measured by directly determining telomere length in sample taken from the patient. Telomere length can be measured, for example, by using standard hybridization techniques, such as fluorescence in situ hybridization (FISH), Quantitative Fluorescent in situ hybridization (Q-FISH), or High Throughput Quantitative Fluorescent in situ hybridization (HT Q-FISH) in a sample taken from the patient. Samples suitable for telomere analysis include blood, urine, or tissue biopsies, such as kidney biopsies.
In a particular embodiment, samples are taken from the patient undergoing treatment throughout the course of the treatment so that both absolute telomere length and the rate of telomere shortening over the course of treatment can be determined. Samples may be taken every day during the course of treatment, or at longer intervals. In one embodiment, samples are taken once a week, once every two week, once every three weeks, once every 4 weeks, once every five weeks, once every six weeks or longer. Comparison of telomere length can be measured by a comparing the proportion of short telomeres in a sample taken from a patient. In one embodiment, the proportion of short telomeres is the fraction of telomeres presenting an intensity below the mean intensity of the sample as measured by a in situ hybridization technique, such as FISH or Q-FISH. In embodiment, the proportion of short telomeres is the fraction of telomeres presenting an intensity 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% or more below the mean intensity of the sample. In one particular embodiment, the proportion of short telomeres is the fraction of telomeres presenting an intensity 50% or more below the mean intensity of the sample.
In another embodiments, the proportion of short telomeres is the fraction of telomeres below a certain length, e.g. 8 kb, 7 kb, 6 kb, 5 kb, or shorter. In one embodiment, the proportion of short telomeres is the fraction of telomeres 8 kb or shorter. In another embodiment, the proportion of short telomeres is the fraction of telomeres 7 kb or shorter. In another embodiment, the proportion of short telomeres is the fraction of telomeres 6 kb or shorter. In another embodiment, the proportion of short telomeres is the fraction of telomeres 5 kb or shorter. In another embodiment, the proportion of short telomeres is the fraction of telomeres 4 kb or shorter. In another embodiment, the proportion of short telomeres is the fraction of telomeres 3 kb or shorter.
In one embodiment, the effectiveness of the treatment is measured by a decrease in the proportion of short telomeres in sample taken from a treated patient suffering from a condition associated with short telomere length, preferably kidney fibrosis, as compared to a control sample. In one embodiment, the proportion of short telomeres in a sample taken from a treated patient is decreased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or greater as compared to a control sample. In one embodiment, the control sample is a sample taken from the same patient prior to the treatment or taken at an earlier stage of the treatment. In another embodiment, the control sample is a sample taken from a patient suffering from the same condition and not provided the treatment.
In a further aspect, the invention is applied to the subject by administering a pharmaceutical composition comprising an effective amount of any one of the gene therapy vectors compatible with the invention described above.
A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
“Composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.
They will usually include components in addition to the active component (such as the gene therapy vector) e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s).
Compositions will generally be administered to a subject in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, although some viral vectors are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other viral vectors are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use. Thus, a composition of the invention may be dried, such as a lyophilised formulation. The composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the composition should be substantially free from (i.e. less than 5 μg/m′i′) mercurial material e.g. thiomersal-free.
To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml e.g. about 10+2 mg/ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290-310 mOsm/kg.
Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range.
The composition may include material for a single administration or may include material for multiple administrations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material.
Compositions of the invention for use in humans are typically administered in a dosage volume of about 0.5 ml, although a half dose (i.e. about 0.25 ml) may be administered to children.
As well as methods of treatment described herein, the invention also provides a nucleic acid sequence encoding a TERT for use in therapy. The invention also provides a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT), for use in a method of therapy and a gene therapy vector comprising a coding sequence for telomerase reverse transcriptase (TERT), for use in a method of therapy. In particular, the therapy may be treating or preventing a condition associated with short telomere length. As described for methods of treatment, the TERT nucleic acid sequence may be the sequence as recited in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment or functional equivalent thereof. The TERT protein may have a sequence as recited in SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment or functional equivalent thereof.
The following embodiments are also derived from the first aspect and included herein:
1. A method of treating a patient with kidney fibrosis associated with short telomere length comprising administering to the patient a nucleic acid vector comprising a coding sequence for telomerase reverse transcriptase (TERT).
2. The method of 1, wherein TERT is encoded by a nucleic acid sequence comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
3. The method of 1 or 2, wherein TERT is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
4. The method of any of 1-3, wherein TERT is encoded by a nucleic acid sequence consisting of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3
5. The method of any of 1-4, wherein TERT comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.
6. The method of any of 1-5, wherein TERT comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.
7. The method of any of 1-6, wherein TERT consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 4.
8. The method of any of 1-7, wherein the nucleic acid sequence encoding TERT is operably linked to a regulatory sequence that drives the expression of the coding sequence.
9. The method of any of 1-8, wherein the vector is a non-integrative vector.
10. The method of any of 1-9, wherein the vector is an adeno-associated virus-based non-integrative vector or a RNA vector.
11. The method of any of 1-10, wherein the vector is an adeno-associated virus-based vector derived from a serotype 9 adeno-associated virus (AAV9).
12. The method of 11, wherein the capsid of the adeno-associated virus-based vector is made of capsid proteins of the serotype 9 adeno-associated virus (AAV9), and the nucleic acid sequence contained in the capsid is flanked at both ends by internal terminal repeats corresponding to serotype 2 adenoassociated viruses.
13. The method of 12, wherein the nucleic acid contained in the capsid comprises a fragment which encodes the amino acid sequence coding for TERT.
14. The method of any of 1-13, wherein the vector comprises a regulatory sequence which is a constitutive promoter.
15. The method of 14, wherein the regulatory sequence is the cytomegalovirus (CMV) promoter.
16. The method of any of 1-15, wherein the kidney fibrosis associated with short telomere length is characterized by mutations in a gene or genes involved in telomere maintenance.
The invention further provides in a second aspect a non-human knock-out animal model characterized in that it exhibits a pathological condition of kidney fibrosis, wherein said non-human animal is obtained or obtainable when a sublethal dose of folic acid is administered to a non-human knock-out animal comprising a deactivation of at least one, preferably both, alleles of the Tert gene. By “deactivation of at least one, preferably both, alleles of the Tert gene” is referred herein to the process in which the sequence of one or both alleles of the Tert gene is altered thereby causing said allele(s) to lose its biological function. The alteration in the sequence of the allele(s) may be by inserting nucleotides into said allele(s) or by partially or totally deleting the nucleotides of said allele(s).
Specifically, the non-human knock-out animal model has a knock-out mutation in the Tert gene, resulting in the knock-out of Tert. The term “knock-out animal” as used herein refers to a non-human animal, preferably a mammal, which carries one or more genetic manipulations leading to deactivation of the Tert gene, either only one allele or preferably both alleles of said gene. Said knock-out mutation of one or both of the alleles of the Tert gene may be present in the animal's germ cells, somatic cells, or in both (i.e., the knock-out mutation of the Tert gene is in all the animal's cells). By “germ cells” is referred herein as the germ cells or gametes (eggs and sperm). By “somatic cells” is referred herein as the cells forming the body of a multicellular organism other than germ cells or gametes.
In a preferred embodiment, said non-human knock-out animal is a rodent, such as a mouse or rat. The male animal founders are referred as G0, while the first, second, third generation are referred to as G1, G2, G3, respectively. Preferably, the non-human knock-out animal is the third generation (G3) or subsequent generations (G4, G5, etc) of the Tert−/− know-out animal lineage. Preferably, the non-human animal model is a G3 Tert−/− mouse. The expression “G3 Tert−/−” refers herein to the third generation of animals, preferably mice, that comprise a deactivation in both alleles of the Tert gene, preferably wherein said deactivation is hereditary, i.e., it is present in the germ cells of the animal. To produce a colony of Tert−/− animals, heterozygous animals Tert+/+ are intercrossed to produce homozygous animals Tert−/− for further phenotyping. The knock-out technology is well known in the art.
Embryonal cells at various developmental stages can be used to introduce genes for the production of knock-out animals. Different methods are used depending on the stage of development of the embryonal cell. Such transfected embryonic stem (ES) cells can thereafter colonize an embryo following their introduction into the blastocoele of a blastocyst-stage embryo and contribute to the germ cells of the resulting chimeric animal. Prior to the introduction of transfected ES cells into the blastocoele, the transfected ES cells can be subjected to various selection protocols to enrich the proportion of ES cells that have integrated into knock-out gene if the knock-out gene provides a means for such selection. Alternatively, PCR can be used to screen for ES cells that have integrated the knock-out.
In addition, retroviral infection can also be used to introduce knock-out genes into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection. Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida. The viral vector system used to introduce the knock-out is typically a replication-defective retrovirus carrying the knock-out. Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells. Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele. Most of the founders will be mosaic for the knock-out gene since incorporation occurs only in a subset of cells, which form the knock-out animal. Further, the founder can contain various retroviral insertions of the knock-out gene at different positions in the genome, which generally will segregate in the offspring. In addition, knock-out genes may be introduced into the germline by intrauterine retroviral infection of the mid-gestation embryo. Additional means of using retroviruses or retroviral vectors to create knock-out animals known to those of skill in the art involve the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos.
Once the founder animals are produced, they can be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. The knock-out animals are screened and evaluated to select those animals having the phenotype of interest. Initial screening can be performed using, for example, Southern blot analysis or PCR techniques to analyze animal tissues to verify that the knock-out gene has taken place. The know-out animals can be identified by assessing the levels of TERT mRNA expression in the tissues of the knock-out animals using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of the suitable tissues can be evaluated immunocytochemically using antibodies specific for Tert protein or with a tag such as EGFP. The knock-out non-human mammals can be further characterized to identify those animals having a phenotype useful in the invention. In particular, knock-out non-human mammals having one or both Tert alleles deactivated can be screened according to the presence of kidney pathology or fibrosis once a sublethal dose of FA has been administered to them.
As shown in the examples below, the a G3 Tert−/− non-human knock-out mouse only becomes a non-human animal model for kidney fibrosis when a sublethal dose of FA has been administered to it. Therefore, it is noted that the non-human animal model according to the second aspect that is characterized in that it is a knock-out for at least one, preferably both of the alleles of the gene coding of the telomerase catalytic subunit Tert, is further characterized for exhibiting a pathological condition of kidney fibrosis upon treatment with a sublethal dose of folic acid. By “sublethal dose” is referred herein as a dose that does not induce kidney fibrosis in wild-type mice. In an embodiment, the sublethal dose is less than 250 mg/kg body weight. Preferably, the dose of FA administered to the non-human animal ranges between 50 and 200 mg/kg, more preferably 50-175 mg/kg, 50-150 mg/kg, 100-150 mg/kg, most preferably of about 125 mg/kg body weight. Preferably, the dose of FA is a dose of up to 200 mg/kg body weight, more preferably of up to 125 mg/kg body weight.
The non-human knock-out animal model for kidney fibrosis is further characterized by having an increased creatinine and BUN levels in the blood upon administration of a sublethal dose of FA. Further, said non-human knock-out animal model presents kidney fibrosis as detected by Masson's trichrome staining and increased renal tubular injury as determined by increased PAS+D staining, as shown in the examples below.
In an embodiment, the dose of FA is administered once, preferably at the start of the study. In another embodiments, the FA is administered several times. In some other embodiments, the FA is administered once per day during several days (such as, once every day, once every two days, once every week, once every two weeks, or once every month). In some embodiments, the dose of FA is administered systemically, preferably intraperitoneally or intravenous.
The non-human knock-out animal to which FA is administered to generate the non-human animal model of the second aspect is between 4-10 weeks of age, preferably between 6-8 weeks of age.
In a third aspect, the invention provides a non-human knock-out animal model for use in studying and/or evaluating evaluate kidney fibrosis associated to the presence of short telomeres, wherein the non-human knock-out animal is as defined in the second aspect or any of its embodiments.
In a fourth aspect, the invention provides methods for producing a non-human knockout animal model for evaluating kidney fibrosis associated to the presence of short telomeres, the method comprising the steps of:
In a fifth aspect, the invention provides a non-human knock-out animal model for use in evaluating kidney fibrosis associated to the presence of short telomeres, wherein said non-human animal is obtained according to the method of the fourth aspect or any of its embodiments.
In a sixth aspect, the invention provides a method of evaluating kidney fibrosis associated to the presence of short telomeres, wherein the method comprises the use of the non-human animal model as defined in the second aspect or any of its embodiments.
In a seventh aspect, invention provides the non-human animal model as defined in the second aspect or any of its embodiments for use in a method for screening compounds to improve, treat or prevent, kidney fibrosis associated to the presence of short telomeres. In a preferred embodiment, the method comprises the steps of:
In an eighth aspect, the invention provides a screening method for a test compound for kidney fibrosis associated to the presence of short telomeres, wherein the test compound is administered to the non-human knock-out animal model as defined in the second aspect or any of its embodiments, wherein any change in life expectancy or in symptoms related to kidney fibrosis associated to the presence of short telomeres are measured and evaluated, and wherein the test compound in which said changes were improved is determined as having a therapeutic effect. Particular embodiments of the screening method of the seventh or eighth aspects include steps wherein the tissues, organs or cells derived from said non-human knock-out animal model are contacted with the test compound, and a change in said tissues, organs or cells is measured and evaluated to determine which test compound caused an improvement and therefore has a therapeutic effect.
The test compounds found to have a therapeutic effect for kidney fibrosis associated to the presence of short telomeres which can be obtained by the screening method disclosed herein in the seventh or eighth aspects, can be used for therapy in patients who have developed or are at risk of developing kidney fibrosis associated to the presence of short telomeres. In an embodiment of the screening methods, the case of the non-human animal model according to the second aspect or any of its embodiment is compared and evaluated with the case of a wild-type mouse or with a non-human animal according to the second aspect not treated with the test compound.
In a ninth aspect, the present invention relates to the use of a non-human animal model as defined in the second aspect or any of its embodiments in a screening method for a test compound for kidney fibrosis associated to the presence of short telomeres. In a preferred embodiment, the method comprises the steps of:
It is noted that the embodiments and definitions included under the second aspect of the present invention also apply to the third, fourth, five, sixth, seventh, eighth and ninth aspects.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
Tert heterozygous mice generated as previously described63 were backcrossed to a >98% C57BL/6 background. Tert+/− mice were intercrossed to generate first generation (G1) homozygous Tert−/− knockout mice. G3 Tert−/− mice were generated by successive breeding of G2 Tert−/− mice. Tert+/+ and G3 Tert−/− male and female mice of pure C57BL/6 background were treated with FA (F7876; Sigma-Aldrich). Male and female G3 Tert−/− mice (aged 7, 27 and 47 weeks) were euthanized to analyze the kidney phenotype for signs of fibrosis and for RNA-seq analysis.
Trf1lox/lox mice64 were crossed with mice expressing CreERT2 recombinase driven by the tamoxifen-inducible ubiquitin C (UBC) promoter65 to generate Trf1lox/lox;hUBC-CreERT2 or Trf1+/+;hUBC-CreERT2 mice. These mice were fed ad libitum with a long-term tamoxifen-containing diet, starting at 10 weeks of age.
All mice were generated and maintained at the Animal Facility of the Spanish National Cancer Research Centre (CNIO) under specific pathogen-free conditions with a 12-h light/dark cycle. Mice were housed in plastic cages containing wood shavings or chipped wood bedding with food and water available ad libitum in accordance with the recommendation of the Federation of European Laboratory Animal Science Associations. All animal procedures were approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare (CBA 03_2019-v2).
A single intraperitoneal injection of FA with different doses (50, 100, 125 and 250 mg kg−1 body weight) in vehicle (0.2 ml of 0.3 mmol l−1 NaHCO3) or vehicle alone was administered to 6- to 8-week-old Tert+/+ and G3 Tert−/− mice. Blood was collected at days 7 and 14 and was analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized at day 14 after FA injection, and the kidneys were collected from FA-treated or vehicle-treated animals and analyzed by immunohistochemistry for kidney fibrosis.
A single intraperitoneal injection of FA with a low dose of 125 mg kg−1 body weight or 0.3 M NaHCO3 (200 ul) was inoculated to 6- to 8-week-old Tert+/+ and G3 Tert−/− mice. Blood collected at days 2, 7 and 14 and was analyzed by VetScan Comprehensive Diagnostic Profile kit. Mice were euthanized at day 14, and kidneys were perfused with cold PBS and harvested.
Kidneys were fixed in 4% formaldehyde and embedded in paraffin. Paraffin sections (5-μm thick) were stained with Masson's trichrome, Sirius red and PAS+D using standard procedures. Percentages of fibrotic areas were quantified using the National Institutes of Health (NIH) ImageJ program.
Primary mouse PTCs were isolated from 10- to 11-week-old Tert+/+ and G3 Tert−/− mice as described previously66. mTert-pBabe-puro was a gift from M. Alvarez and J. Bidwell (Addgene plasmid no. 36413). Retroviral plasmid vectors (mTert-pBabe-puro) and the packaging plasmids (PLC Eco and pCMV-VSV-G) were co-transfected into the packaging cell line 293T. Viral supernatants were collected 48 h later, centrifuged to remove cell debris, strained through 0.45-μm filters (Millipore) and used to infect isolated PTCs from Tert+/+ and G3 Tert−/− mice. Stable cell lines were selected with 2 μg ml−1 puromycin on day 10 until day 14. Cells were collected for RNA extraction and immunofluorescence.
Kidney tissues were fixed in 4% formaldehyde and embedded in paraffin. Paraffin-embedded kidney tissue sections at a thickness of 5-7 μm were subjected to immunohistochemical staining. Primary antibodies (and their dilutions) were: rat monoclonal to p21 (HUGO-291H/B5; CNIO histopathology core unit; 1:400), rat monoclonal to p53 (POE316A/E9; CNIO histopathology core unit; 1:400), rat monoclonal to p21 (HUGO-291H/B5; CNIO histopathology core unit; 1:400) rat monoclonal to CD8a (AM-OTO94A; CNIO histopathology core unit), mouse monoclonal to phospho-Histone H2AX (Ser 139; 05-636; Millipore; 1:400), mouse monoclonal to E-cadherin (610182; BD Biosciences; 1:400), rat monoclonal to F4/80 (MCA497; AbD Serotec; 1:400), rabbit polyclonal activated caspase-3 (9661; Cell Signaling Technology; 1:400), rabbit monoclonal to CD3e (99940; Cell Signaling Technology; 1:400), rabbit monoclonal to CD4 (25229; Cell Signaling Technology; 1:400), rabbit polyclonal to Collagen type VI (ab6588; Abcam; 1:400), rabbit polyclonal to fibronectin (ab2413; Abcam; 1:400) and rabbit polyclonal to Sox9 (AB5535; Millipore; 1:400).
For immunofluorescence, mouse monoclonal antibody to α-SMA-Cy3 (C6198; Sigma; 1:400), rabbit monoclonal to vimentin (5741; Cell Signaling Technology; 1:200), rabbit monoclonal to Ki67 (12202; Cell Signaling Technology; 1:400), rabbit polyclonal TGFβ (3711S; Cell Signaling Technology; 1:200), rabbit polyclonal SNAIL+SLUG (ab180714; Abcam; 1:200) and rat monoclonal E-cadherin (DECMA-1; ab11512, Abcam; 1:200) were used. Images were obtained using a confocal ultra-spectral microscope (Leica TCS-SP5). The percentages of positively stained areas by immunohistochemistry and immunofluorescence were quantified using NIH ImageJ (v1.52n).
Total RNA was isolated from kidney tissues and PTCs using TRIzol reagent (Takara), according to the manufacturer's instructions. cDNA was synthesized with 1 μg of total RNA, cDNA synthesis mix (BioMake), and oligo-dT primers. Gene expression was measured by a real-time PCR assay (BioMake) and a 7900HT real-time PCR system (Applied Biosystems). The relative amount of mRNA to the internal control was calculated as 2ΔCT, in which ΔCT=ΔCTexperimental−ΔCTcontrol. Genes and primers are listed in Table 2 (F: forward primer; R: reverse primer).
For RNA-seq experiments, total RNA samples (300 ng) were used. RNA quality scores were 6.3 on average (range 4.7-8.0) when assayed on a PerkinElmer LabChip analyzer. Sequencing libraries were prepared with the QuantSeq 3′ mRNA-Seq Library Prep Kit (FWD) for Illumina (Lexogen; 015), following the manufacturer's instructions. Library generation was initiated by reverse transcription with oligo-dT priming, and a second-strand synthesis was performed from random primers by a DNA polymerase. Primers from both steps contained Illumina-compatible sequences. cDNA libraries were purified, applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NextSeq 550 (with v2.5 reagent kits), following the manufacturer's protocols. Read adaptors and poly(A) tails were removed with the command ‘bbduk.sh’, following the Lexogen recommendations. Processed reads were analyzed with the Nextpresso pipeline67 as follows. Sequencing quality was checked with FastQC v0.11.7 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to the mouse reference genome (GRCm38) with TopHat (v2.0.10)68 using Bowtie (v1.0.0)69 and SAMtools (v0.1.19)70 (--library-type fr-secondstrand in TopHat), allowing three mismatches and twenty multihits. Read counts were obtained with HTSeq-count (v0.6.1)71 using the mouse gene annotation from GENCODE (GRCm38; vM20 Ensembl 95). GSEAPreranked72 was used to perform GSEA for several gene signatures on a preranked gene list, setting 1,000 gene-set permutations. Only those gene sets with significant enrichment levels (FDR q value <0.25) were considered.
Q-FISH determination on paraffin-embedded tissue sections was performed as described previously41. After deparaffinization, tissues were postfixed in 4% formaldehyde for 5 min, washed three times for 5 min each in PBS and incubated at 37° C. for 15 min in pepsin solution (0.1% porcine pepsin, Sigma; 0.01 M HCl, Merck). After another round of washes and fixation as mentioned above, slides were dehydrated in an ethanol series (70%, 90% and 100%; 5 min each). After 10 min of air drying, 30 l of telomere probe mix (10 mM Tris-CI (pH 7), 25 mM MgCl2, 9 mM citric acid, 82 mM Na2HPO4, 70% deionized formamide (Sigma), 0.25% blocking reagent (Roche) and 0.5 μg ml−1 telomeric PNA probe (Panagene) were added to each slide. A coverslip was added and slides were incubated for 3 min at 85° C., and for a further 2 h at room temperature in a wet chamber in the dark. Slides were washed twice for 15 min each in 10 mM Tris-CI (pH 7), 0.1% BSA in 70% formamide under vigorous shaking, then three times for 5 min each in TBS 0.08% with Tween 20 and then incubated in a DAPI bath (4 μg ml−1 in PBS; Sigma) before mounting samples in Vectashield medium (Vector). Confocal images were acquired as stacks every 1 μm for a total of 5 μm using a Leica SP5-MP confocal microscope, and maximum projections were done with LAS-AF software. Telomere signal intensity was quantified using Definiens software.
No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported previously446. Mice were randomly allocated within the groups, and investigators were blinded to group allocation. Our sample sizes corresponded to the number of mice used for each experiment, as indicated. The quantitative analyses of immunohistochemistry stainings were performed in the whole scanned kidney section comprising 10-15 areas. For immunofluorescence analysis, 10-20 images were collected from each individual. Data distribution was assumed to be normal, but this was not formally tested. No data points or mice were excluded from the analyses. Results of statistical analyses are expressed as means±s.e.m. Statistical analyses of immunohistochemical/immunofluorescence quantifications and of qPCR and Q-FISH analyses were performed using one-way ANOVA with post hoc Tukey's test in Prism (GraphPad). Two-way ANOVA with post hoc Bonferroni's test was used to analyze the blood and urine parameters. Statistical significance was defined as P<0.05.
A Mouse Model of Kidney Fibrosis is Associated with Short Telomeres
To test the involvement of short telomeres in kidney fibrosis, we first analyzed the kidneys of wild-type mice, as well as mice lacking the telomerase catalytic subunit, Tert, that were bred for three generations (G3) to induce the presence of very short telomeres, that is, G3 Tert−/− mice24 (
The incidence of kidney fibrosis increases with age; therefore, it is likely to be caused by the combination of molecular and cellular aging events, such as the presence of short telomeres14 together with exogenous damage to the kidney. Thus, we next challenged the kidneys of Tert+/+ and G3 Tert−/− mice with FA, previously described to induce kidney fibrosis at a high dose of 250 mg kg−1 body weight27. FA-induced nephropathy is widely used to study interstitial kidney fibrosis4,27,28. In particular, FA administered intraperitoneally in mice leads to rapid appearance of FA crystals in tubules, followed by severe nephrotoxicity.
To this end, we first subjected Tert+/+ and G3 Tert−/− mice aged 8-9 weeks to increasing doses of FA (50, 100, 125 and 250 mg kg−1 body weight;
Indeed, we treated G3 Tert−/− mice with the same doses of FA and observed that, even after the 125 mg kg−1 dose, the kidneys presented a pale color (
Severe Kidney Dysfunction in Telomerase-Deficient Mice Treated with a Sublethal Dose of Folic Acid
To address the role of short telomeres in kidney fibrosis, we subjected Tert+/+ and G3 Tert−/− mice at 8-9 weeks of age to a dose of 125 mg kg−1 body weight of FA, which does not induce fibrosis in wild-type mice (
We also examined BUN and creatinine in all mouse cohorts. Administration of a high dose of FA is known to induce a transient elevation of BUN and creatinine levels at 48 h after injection followed by subsequent renal dysfunction accompanied with interstitial fibrosis29. Two days after the injection of a low dose of FA (125 mg kg−1 body weight), however, both the untreated and FA-treated wild-type mice (Tert+/+) mice showed normal BUN and creatinine levels, indicating normal kidney function (
bp < 0.001 vs. FA-treated Tert+/+,
cp < 0.001 vs. G3Tert−/−
dp < 0.01 vs. Tert+/+,
ep < 0.01 vs. FA-treated Tert+/+,
fp < 0.01 vs. G3Tert−/−
gp < 0.05 vs. Tert+/+;
hp < 0.05 vs. FA-treated Tert+/+,
ip < 0.05 vs. G3Tert−/−
Telomerase-Deficient Mice Show Collagen Deposition and Activated Myofibroblasts in the Kidney after a Sublethal Dose of Folic Acid
It has been previously shown that FA-induced renal injury concurs with development of segmental interstitial fibrotic lesions approximately 2 weeks after treatment37. In agreement with this, at 14 d after treatment, we observed interstitial fibrotic areas as indicated both by Masson's trichrome and Sirius red staining in the kidneys of FA-treated G3 Tert−/− mice, while no fibrosis was detected in the kidneys from G3 Tert−/−, Tert+/+ and FA-treated Tert+/+ mice (
EMT involves a change from the apical-basolateral polarity of the epithelial cells to the front-rear polarity of the mesenchymal cells and the expression of mesenchymal markers, such as fibroblast-specific protein-1, vimentin, N-cadherin and α-SMA. These changes induce enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and increased production of ECM components38. To demonstrate that the proliferating cells observed in FA-treated G3 Tert−/− mice are indeed EMT cells and not tubular cells, we performed double immunofluorescence staining for Ki67 and α-SMA. We found that 36% of proliferating cells were myofibroblasts (α-SMA+Ki67+) in FA-treated G3 Tert−/− mice compared to untreated G3 Tert−/− mice (
Increased Kidney Apoptosis and Senescence in Telomerase-Deficient Mice after a Sublethal Dose of Folic Acid
Short telomeres have been shown by us and others to induce a persistent DNA damage response at chromosome ends, leading to cell cycle arrest or apoptosis39,40. In particular, short telomeres induce the p53 and p21 cell cycle inhibitors22,41. Interestingly, p21 is known to be an activator of TGFβ37, thus providing a potential mechanism by which short telomeres, even in the absence of fibrosis, may contribute to activation of EMT programs.
In this regard, we studied whether kidney dysfunction and increased fibrosis in G3 Tert−/− mice treated with FA was accompanied by increased cellular senescence or apoptosis, two well-known cellular responses to telomere dysfunction with aging14. We observed that FA treatment induced a significant increase in levels of CC3 (apoptosis marker), p21 and p53 cell cycle inhibitors, and γ-H2AX (DNA damage marker) in G3 Tert−/− mice, compared to undetectable levels in FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (
Next, we set to study the pathways by which short telomeres induce kidney injury. The kidney injury molecule-1 (KIM-1, also known as hepatitis A virus cellular receptor (HAVCR) 1; encoded by the gene Havcr1) is a type 1 transmembrane protein that is undetectable in healthy kidneys but greatly induced after injury42, localizing to the apical surface of the surviving proximal tubular cells (PTCs)43. Inflammation is proposed as a key driver of kidney fibrosis42, and the neutrophil gelatinase-associated lipocalin (NGAL) gene product (lipocalin-2 (Lcn2) or siderocalin)44 is induced during acute kidney injury (AKI)44. Thus, we used Havcr1 and Lnc2 as biomarkers of CKD due to tubulointerstitial damage. We found that Havcr1 and Lcn2 were upregulated by 134- and 206-fold, respectively (
Recruitment of macrophages in tissue fibrosis is known to involve TGFβ, also important for the EMT process45 associated with kidney fibrosis46. Thus, we next sought to determine whether the presence of short telomeres is associated with changes in the expression of genes involved in EMT. To this end, we studied the expression of EMT and EMT-related pathways such as TGFβ signaling, a major environmental stimulus that induces EMT in adult epithelia, by performing RNA sequencing (RNA-seq) in the kidneys of 10-week-old Tert+/+ and G3 Tert−/− mice either untreated or treated with low-dose FA (125 mg kg−1 body weight). Gene-set enrichment analysis (GSEA) of untreated Tert+/+ versus untreated G3 Tert−/− mice showed that the EMT (normalized enrichment score (NES)=3.37) and the TGFβ (NES=1.9) pathways were upregulated in telomerase-deficient mice compared to wild-type mice (
Upregulation of Key EMT Transcription Factors in Telomerase-Deficient Mice with Short Telomeres
TGFβ1-induced EMT is mediated by ZEB1 and SNAIL in a Smad-dependent manner45. TWIST, SNAIL and ZEB1 are transcription factors that regulate the EMT transcriptional program. Activation of Twist, Snail or Zeb1 is sufficient to induce a mesenchymal phenotype4. Thus, we set to determine the mRNA expression levels of Tgfb1, Snail1, Snail2, Twist1, Zeb1 and Zeb2 by PCR with reverse transcription (RT-PCR). We observed enhanced expression of Tgfb1 by 32-fold in FA-treated G3 Tert−/− mice compared to other mouse cohorts (
To understand how progenitor and stem cells may be affected by short telomeres during FA-induced kidney fibrosis, we studied the expression of sex determining region Y box 9 (Sox9), Wilms' tumor (Wt1), paired box 2 (Pax2), spalt like transcription factor 2 (Sall2), activin A receptor type 2B (Acvr2b, a Tgfb superfamily member) and Klotho (Kl). SOX9 is known to be important in kidney development in mouse48 and human49 studies and required for kidney fibrosis50. This precedes the expression of Wt1, Pax2 and NOTCH signaling within the epithelium. Wt1 maintains a switch between the mesenchymal and epithelial cell states and is needed to induce mesenchymal-to-epithelial transformation (MET), as well as playing a key role in the progression of nephrogenesis5l. PAX2, a nuclear transcription factor, is associated with MET and it is required for kidney cell differentiation52. Re-expression of Wt1 and Pax2 in the tubular epithelial cells plays an important role in the promotion of EMT, and there may be therapeutic value in silencing Pax2 and Wt1 to prevent or reverse kidney fibrosis53. In this regard, we observed a significant increase in the transcript levels of Sox9, Pax2, Wt1 and Acvr2b expression in Tert−/− mice exposed to FA compared to all other groups, suggesting that under those conditions short telomeres sensitized the kidneys to undergo an EMT (
Persistent expression of NOTCH signaling pathways within the epithelium results in interstitial fibrosis57. NOTCH is a strong regulator of SNAIL1 and SNAIL2 (ref. 58). Thus, we analyzed the expression of Notch receptors (Notch1, Notch2 and Notch3), Notch ligand Jagged 1 (Jag1) and mitochondrial transcription factor A (Tfam) as a direct Notch target59 important for kidney function. We found a threefold increase in Notch1 and Notch2 and a fourfold increase in Notch3 and Jag1 in FA-treated G3 Tert−/− mice compared to FA-treated Tert+/+ and untreated Tert+/+ mice and G3 Tert−/− mice (
Trf1 Deletion-Induced Telomere Dysfunction Triggers Kidney Fibrosis Associated with Activation of EMT
To assess the contribution of dysfunctional telomeres to the induction of kidney fibrosis, we used a second model of telomere dysfunction by deleting TRF1, one of the components of the shelterin telomere protective complex. In particular, we used a mouse model previously described by us60 in which treatment with tamoxifen led to deletion of Trf1 in all kidney cells (
Telomerase Overexpression Rescues EMT Changes Associated with Short Telomeres
To further explore a contribution of short telomeres in the activation of EMT programs in the kidney, we addressed whether they could be rescued by expression of the catalytic subunit of telomerase or TERT61. To this end, we first isolated kidney epithelial cells, in particular PTCs, from 10- to 11-week-old Tert+/+ and G3 Tert−/− mice. PTCs were transduced with either an empty vector or with a vector expressing Tert (Methods). Telomerase overexpression was confirmed by qPCR (
EMT is a cellular plasticity process by which epithelial layers lose their integrity concomitant with a loss of cell polarity and cell-to-cell interactions mediated by loss of E-cadherin3. The resulting cells express mesenchymal properties, including expression of vimentin and α-SMA. EMT is orchestrated by a number of transcription factors (EMT-TFs) and has key roles both during normal development3 and in pathological conditions such as cancer and various tissue fibrotic diseases, including kidney fibrosis4,62.
As both cancer and tissue fibrosis have been associated with aging, it is of particular relevance to understand how known molecular and cellular mechanisms of aging14 may impact the expression of EMT-TFs and the origin of EMT pathological processes.
Accumulation of short and dysfunctional telomeres associated with cell division during the life span of an organism is considered one of the primary hallmarks of aging as it triggers persistent DNA damage that it is sufficient to impair the regenerative capacity of adult stem cell compartments10,13. Indeed, accelerated telomere shortening owing to telomerase deficiency both in mice24,25 and humans8 leads to premature loss of the regenerative capacity of tissues, including development of tissue fibrosis, of which pulmonary fibrosis is one of the most prevalent8,22.
Here, we generated a mouse model of fibrosis in the kidney associated with short telomeres by challenging telomerase-deficient mice with short telomeres to a low, sublethal dose of FA, a damaging agent to the kidney, which does not induce fibrosis in similarly treated wild-type controls. Telomerase-deficient mice treated with a sublethal dose of FA show all the hallmarks of the human disease including severe kidney dysfunction, as indicated by elevated blood levels of creatinine and urea. In addition, mice with dysfunctional telomeres owing to deletion of Trf1 encoding for a shelterin protein spontaneously develop kidney fibrosis, highlighting the importance of proper telomere function in the protection of fibrotic pathologies. Thus, the new mouse models generated here represent good tools to understand the role of short and dysfunctional telomeres and resulting DNA damage in the molecular events associated with fibrosis.
Interestingly, we find here that short telomeres lead to changes in the expression of genes involved in EMT, although these changes were not sufficient to induce kidney fibrosis. These changes were further exacerbated in the telomerase-deficient mice treated with FA that develop kidney fibrosis. This is supported by increased expression of Snail1, Snail2, Zeb1, Zeb2 and Twist1 transcription factors that results in epithelial cells turning into myofibroblasts. Myofibroblasts deposit ECM containing collagen leading to the development of kidney fibrosis.
Supporting evidence that short telomeres contribute to EMT changes, we show that expression of the mouse telomerase TERT catalytic subunit and the subsequent elongation of telomeres are sufficient to revert EMT programs and to restore an epithelial phenotype in kidney cells in culture.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/051505 | 1/24/2022 | WO |
