Patent Application
20200230207
The present invention relates to an activator of beclin 1-Vps 34 complex for use in the treatment and/or prevention of a bone growth disorder. The activator may be a polypeptide, a polynucleotide, a vector, a host cell or a small molecule. In particular the activator may be a Beclin 1 peptide or a fragment or a derivative thereof, a mTORC1 inhibitor or a BH3 mimetic. The present invention also relates to pharmaceutical composition comprising said activator.
Bones in different parts of the skeleton develop through two distinct processes, intramembranous ossification and endochondral ossification. Intramembranous ossification occurs in the flat bones of the skull and involves direct differentiation of embryonic mesenchymal cells into the bone-forming osteoblasts. Endochondral ossification is responsible for the initial bone development from cartilage, in utero and infants; furthermore it is an essential process during formation of long bones, for the longitudinal growth of long bones and for the natural healing of bone fractures.
Endochondral ossification begins when mesenchymal cells differentiate into chondrocytes, which secrete the various components of cartilage extracellular matrix (ECM), including collagen type II and the proteoglycan aggrecan, and which form a cartilage template for future bone. Ossification of the cartilage model is preceded by chondrocytes proliferation and hypertrophy. The primary centre of ossification, wherein blood vessels, osteoclasts, bone marrow and osteoblast precursors invade the model, expands towards the ends of the cartilage model, as the osteoclasts remove cartilage ECM and osteoblasts deposit bone on cartilage remnants. In long bones, a secondary ossification centre subsequently forms at each end of the cartilage model, leaving a cartilaginous growth plate between the primary and secondary ossification centres. Chondrocytes arranged into columns form the growth plate.
The growth plate (also called epiphyseal plate or physis) is a hyaline cartilage plate in the metaphysis at each end of a long bone. The plate is found in children and adolescents; in adults, who have stopped growing, the growth plate is replaced by the epiphyseal line. The growth plate is responsible for longitudinal growth of bones. Skeletal maturity occurs when the expanding primary centre of ossification meets the secondary centre of ossification.
Chondrocyte's rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in the growth plates mediate bone elongation.
Collagens are major structural components of the ECM. Type II collagen (Col2), also called cartilage collagen, is the major collagen synthesized by chondrocytes.
Type II collagen is comprised of 3 alpha-1(II) chains. These are synthesized in the chondrocytes of the growth plate as larger procollagen (PC2) chains, which contain N- and C-terminal amino acid sequences called pro-peptides. After secretion into the extracellular matrix, the pro-peptides are cleaved, forming the mature type II collagen molecule.
As the hypertrophic chondrocytes degenerate, osteoblasts ossify the remains to form new bone. Thus, the growth plate chondrocyte plays multiple important roles during its lifespan. It constructs the transient growth plate tissue, which has the necessary capacity to move in space through continued self-renewal and localized degradation, but simultaneously maintains the mechanical stability of the growing bone.
Defects in the development and maintenance of the growth plates lead to disorders of the bone growth.
Several bone diseases are associated to defects of the collagens, in particular of type II collagen, in particular those due to mutations of COL2A1 gene, coding for the pro-alpha chain of type II collagen (Kuivaniemi et al., 1997). Diseases associated to defects of type II collagen include: achondrogenesis Type II (due to mutation in the type II procollagen gene, leading to abnormal pro-alpha-1(II) chain and impaired assembly and/or folding of type II collagen), platyspondylic skeletal dysplasia, Torrance type, Hypochondrogenesis, Spondyloepiphyseal Dysplasia Congenita (SED), Spondylometaphyseal dysplasia (SMD), Kniest Dysplasia, Stickler Syndrome, Type I, Osteoarthritis Associated with Chondrodysplasia, Avascular Necrosis of the Femoral Head and Legg-Calve-Perthes Disease, Otospondylomegaepiphyseal Dysplasia, Strudwick type of spondyloepimetaphyseal dysplasia, Multiple epiphyseal dysplasia with myopia and conductive deafness, Spondyloperipheral dysplasia, Czech dysplasia.
The most common bone growth disorder is achondroplasia. Achondroplasia is the most common cause of dwarfism. Achondroplasia family is characterized by a continuum of severity ranging from mild (hypochondroplasia, HCH; OMIM:146000) and more severe forms (achondroplasia) to lethal neonatal dwarfism (thanatophoric dysplasia, TD; OMIM:187600). The condition occurs in 1 in 15,000 to 40,000 newborns. Affected individuals exhibit short stature caused by rhizomelic shortening of the limbs, characteristic facies with frontal bossing and midface hypoplasia, exaggerated lumbar lordosis, limitation of elbow extension, genu varum, and trident hand.
Two specific mutations in the FGFR3 gene are responsible for almost all cases of achondroplasia. These mutations cause the FGFR3 protein to be overly active, which interferes with skeletal development and leads to the disturbances in bone growth seen with this disorder.
Dominant mutations in the FGFR3 gene affect predominantly bones that develop by endochondral ossification, whereas dominant mutations involving FGFR1 (OMIM:136350) and FGFR2 (OMIM:176943) principally cause syndromes that involve bones arising by membranous ossification.
Other FGFR3 associated diseases include: thanatophoric dysplasia types 1 and 2 and SADDAN (severe achondroplasia-developmental delay-acanthosis nigricans).
Hypochondroplasia is a form of short-limbed dwarfism. This condition affects the conversion of cartilage into bone (a process called ossification), particularly in the long bones of the arms and legs. Hypochondroplasia is similar to achondroplasia, but the features tend to be milder. About 70 percent of all cases of hypochondroplasia are caused by mutations in the FGFR3 gene. The incidence of hypochondroplasia is unknown. Researchers believe that it may be about as common as achondroplasia, which occurs in 1 in 15,000 to 40,000 newborns. More than 200 people worldwide have been diagnosed with hypochondroplasia.
Evidences indicate that activated FGFR3 is targeted for lysosomal degradation and that activating mutations found in patients with achondroplasia and related chondrodysplasias disturb this process, leading to recycling of activated receptors and amplification of FGFR3 signals (Cho et al., 2004).
Fibroblastic growth factors (FGF) are a family of polypeptides that are involved in numerous developmental processes including embryonic and skeletal development. The function of FGFs is dependent on the spatial and temporal expression of FGF receptors.
FGF18 is an important mediator for skeletal development. Murine Fgf18 binds primarily to FGFR3; furthermore, it binds to FGFR1 in chondrocytes. Inhibition of chondrocyte proliferation and differentiation by FGF18 stimulation in embryos has been previously reported (Kapadia et al., 2005). Further studies indicate that FGF18 positively regulates osteogenesis and negatively regulates chondrogenesis (Ohbayashi, 2002). The activation of FGFR3 has been reported to inhibit the proliferation and differentiation of growth plate chondrocytes (Naski et al., 1998). On the contrary, FGF18 has been shown to have positive effects on chondrocytes in other cartilaginous tissues apart from the growth plate and it has recently been shown that intra articular injection of FGF18 can stimulate the repair of damaged cartilage in a rat model of osteoarthritis (Moore et al., 2005).
Both FGFR3 and FGF18 knockout mice reveal the same phenotype of long bones during embryonic development. All Fgf18−/− mice express skeletal abnormalities including curved radius and tibia and some mice show incomplete development of the fibula. Embryos are approximately 10-15% smaller than the wild type (Liu et al., 2002). The length of the long bone however is considerably smaller in FGF18−/− mice, in comparison to the wild-type, than for FGFR3−/− mice. This difference implies that other signaling pathways, such as FGF18 interaction with other FGF receptors, may be involved in osteogenesis of developing long bone (Ohbayashi et al., 2002).
A genome wide association study, showing that FGFR4 sequence variations may influence human height is described by Lango Allen, H. et al.
Defects in the bone growth are also associated to several Lysosomal storage disorders (LSDs).
Lysosomal storage disorders affect multiple organs including the skeleton. LSDs are a group of approximately 70 inherited diseases characterized by lysosomal dysfunction and neurodegeneration. Although individually rare, the lysosomal storage disorders (LSDs) as a group have a frequency of about 1:8000 live births, making this disease group a major challenge for the health care system. So far, mutations in more than 20 genes encoding for lysosomal proteins cause defects in bone growth and development.
LSDs with prominent skeletal symptoms include type 1 and type 3 Gaucher disease, the mucopolysaccharidoses, multiple sulfatase deficiency, mucolipidosis type II and III, galactosidosis, mannosidosis (alpha and beta), fucosidosis and pycnodysostosis (Clarke and Hollak, 2015).
The mucopolysaccharidosis (MPS) syndromes are lysosomal storage diseases with an overall incidence of about 1:25000. Skeletal manifestations are often the presenting symptom(s) for patients with MPS I, II, IV, VI, VII and IX. Disease symptoms include alteration of linear bone growth, morphologic abnormalities of bone shape and structural as well as functional abnormalities in articular cartilage. Alteration of linear bone growth leading to proportionate short stature is a characteristic feature of all severely affected MPS I, II, IV, VI and VII patients, who show relatively normal linear growth in the first 18 months of life followed by a period of impaired growth with little or no further growth after the age of 8 years.
Hurler and Scheie syndromes represent phenotypes at the severe and mild ends of the MPS I clinical spectrum, respectively, and the Hurler-Scheie syndrome is intermediate in phenotypic expression. Length is often normal until about 2 years of age when growth stops; by age 3 years height is less than the third percentile. The long tubular bones show diaphyseal widening with small, deformed epiphyses. Phalanges are bullet-shaped with proximal pointing of the second to fifth metacarpals. Hurler syndrome is characterized by skeletal abnormalities, cognitive impairment, heart disease, respiratory problems, enlarged liver and spleen, characteristic facies and reduced life expectancy. The prevalence of the Hurler subtype of MPS 1 is estimated at 1/200,000 in Europe. Scheie syndrome is characterized by skeletal deformities and a delay in motor development. Prevalence of Scheie syndrome is estimated at 1/500,000.
Mucopolysaccharidosis type 2 (MPS 2) is a lysosomal storage disease leading to a massive accumulation of glycosaminoglycans and a wide variety of symptoms including distinctive coarse facial features, short stature, cardio-respiratory involvement and skeletal abnormalities. It manifests as a continuum varying from a severe to an attenuated form without neuronal involvement. Prevalence at birth in Europe is 1/166,000. It is an X-linked recessive disorder; very rare cases of female presentation have been reported.
Mucopolysaccharidosis type 4 (MPS IV) is a lysosomal storage disease belonging to the group of mucopolysaccharidoses, and characterised by spondylo-epiphyso-metaphyseal dysplasia. It exists in two forms, A and B. Prevalence is approximately 1:250000 for type WA but incidence varies widely between countries. MPS IVB is even rarer. MPS IVA is characterized by intracellular accumulation of keratan sulfate and chondroitin-6-sulfate. Key clinical features include short stature, skeletal dysplasia, dental anomalies, and corneal clouding.
Mucopolysaccharidosis type 6 (MPS VI) is a lysosomal storage disease with progressive multisystem involvement, associated with a deficiency of arylsulfatase B (ASB or ARSB) leading to the accumulation of dermatan sulfate. Birth prevalence is between 1 in 43,261 and 1 in 1,505,160 live births. Prevalence: 1-9/100000. Mucopolysaccharidosis type VI results from a deficiency of arylsulfatase B. Clinical features and severity are variable, but usually include short stature, hepatosplenomegaly, dysostosis multiplex, stiff joints, corneal clouding, cardiac abnormalities, and facial dysmorphism. Intelligence is usually normal.
Mucopolysaccharidosis type 7 (MPS VII or Sly syndrome) is a very rare lysosomal storage disease belonging to the group of mucopolysaccharidoses, resulting from a deficiency of β-glucuronidase (GUSB). Less than 40 patients with neonatal to moderate presentation have been reported since the initial description of the disease by Sly in 1973. However, the frequency of the disease may be underestimated as the most frequent presentation is the antenatal form, which remains underdiagnosed. Prevalence is lower than 1:1,000,000. MPS VII is characterized by the inability to degrade glucuronic acid-containing glycosaminoglycans. The phenotype ranges from severe lethal hydrops fetalis to mild forms with survival into adulthood. Most patients with the intermediate phenotype show hepatomegaly, skeletal anomalies, coarse facies, and variable degrees of mental impairment. Currently, MPS VII lacks an efficient treatment.
Multiple sulfatase deficiency (MSD) is an autosomal recessive inborn error of metabolism resulting in tissue accumulation of sulfatides, sulfated glycosaminoglycans, sphingolipids, and steroid sulfates. The enzymatic defect affects the whole family of sulfatase enzymes; thus, the disorder combines features of metachromatic leukodystrophy and of various mucopolysaccharidoses. Affected individuals show neurologic deterioration with mental retardation, skeletal anomalies, organomegaly, and ichthyosis.
Gaucher disease (GD) is a lysosomal storage disorder encompassing three main forms (types 1, 2 and 3), a fetal form and a variant with cardiac involvement. The prevalence is approximately 1/100,000. GD type 1 (90% of cases) is the chronic and non-neurological form associated with organomegaly (spleen, liver), bone anomalies (pain, osteonecrosis, pathological fractures) and cytopenia. GD is due to mutations in the GBA gene (1q21) that codes for a lysosomal enzyme, glucocerebrosidase, or in very rare cases the PSAP gene that codes for its activator protein (saposin C). The deficiency in glucocerebrosidase leads to the accumulation of glucosylceramidase (or beta-glucocerebrosidase) deposits in the cells of the reticuloendothelial system of the liver, the spleen and the bone marrow (Gaucher cells). Formal diagnosis of the disease is determined by the measurement of glucocerebrosidase levels in circulating leukocytes. Genotyping confirms the diagnosis.
Current treatments for LSDs are enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplantation. However, effects of these interventions on skeletal disease manifestations are less well established and outcomes are highly dependent on disease burden at treatment initiation. Furthermore, the efficacy of these therapeutic strategies has several major limitations, such as the difficulty of reaching particular tissues such as the skeleton. Indeed, gene therapy approaches in different MPS animal models showed very little efficacy on bone defects (Ferla R et al., 2014, Stevenson D A and Steiner R D, 2013).
Although orthopedic surgery and neurosurgery are important components of care for MPS patients this approach to therapy is largely symptomatic and thus does not alter the primary underlying skeletal pathology. Therapies directed towards the primary metabolic block that have been utilized in the MPSs include bone marrow transplantation and enzyme replacement therapy.
The main treatment option for short stature, e.g. in achondroplasia patients, is administration of recombinant growth hormone (rGH). Recently a phase II study started for evaluating the use of BMN 111, a 39 amino acid analog of C-type natriuretic peptide (CNP), for the treatment of achondroplasia.
Improved treatments that target skeletal diseases are however still needed.
The inventors have surprisingly identified dysregulation of endocytic trafficking and autophagy as a target for treating bone growth disorders.
Autophagy is an essential cellular process that consists of selective degradation of cellular components. There are at least three different types of autophagy described: macroautophagy (also referred to as autophagy), microautophagy and chaperone mediated autophagy. The initial step of autophagy is the surrounding and sequestering of cytoplasmic organelles and proteins within an isolation membrane (phagophore). Potential sources for the membrane to generate the phagophore include the Golgi complex, endosomes, the endoplasmic reticulum (ER), mitochondria and the plasma membrane (Kang et al., 2011).
The nascent membranes are fused at their edges to form double-membrane vesicles, called autophagosomes. Autophagosomes undergo a stepwise maturation process, including fusion with acidified endosomal and/or lysosomal vesicles, eventually leading to the delivery of cytoplasmic contents to lysosomal components, where they fuse, then degrade and are recycled.
Autophagy depends on Atg5/Atg7, it is associated with microtubule-associated protein light chain 3 (LC3) truncation and lipidation, and may originate directly from the ER membrane and other membrane organelles. Furthermore, recent study has identified a Atg5/Atg7-independent pathway of autophagy. This pathway of autophagy was not associated with LC3 processing but appeared to involve autophagosome formation from late endosomes and the trans-Golgi.
Beclin 1 (NP_003757) is the mammalian ortholog of yeast Atg6/Vps30 and it is required for Atg5/Atg7-dependent and -independent autophagy. It forms a protein complex with the class III phosphatidylinositol 3-kinase (PI3KC3)Vps34 (NP_001294949.1; NP_002638.2) and with Vps15 (NP_055417). Beclin 1 encodes a 450 amino acid protein with a central coiled coil domain. Within its N-terminus, it contains a BH3-only domain, which mediates binding to anti-apoptotic molecules such as Bcl-2 and Bcl-xL. The most highly conserved region, referred to as the evolutionarily conserved domain (ECD), spans from amino acids 244-337 and is important for its interaction with Vps34.
The Beclin 1/Vps34 complex (also known as class III phosphatidylinositol 3-kinase complex) is a multivalent trafficking effector that regulates autophagosome formation, including the nucleation of the phagophore at the endoplasmic reticulum (autophagic vesicle nucleation) and autophagosomes maturation.
Furthermore, the Beclin 1/Vps34 complex promotes endocytic trafficking (McKnight N C et al., 2014; Levine B et al., 2015).
Besides Vps15, the complex has numerous other binding partners, including Atg14L (another core autophagy protein), UVRAG (a protein that functions in autophagosomal maturation and endocytic maturation) and Ambral (a positive regulator of the Beclin 1/Vps34 complex). In addition, Beclin 1 has been reported to interact with certain receptors and immune signaling adaptor proteins, including the inositol 1, 4, 5-triphosphate receptor (IP3R), the estrogen receptor, MyD88 and TRIF, and nPIST, as well as certain viral virulence proteins such as HSV-1 ICP34, KSHV vBcl-2, HIV-1 Nef, and influenza M2. A further binding partner is Rubicon, which however is a negative regulator of the Beclin 1/Vps34 complex.
Activation of a Beclin 1/Vps34 complex thus induces autophagy in a cell and/or promotes endocytic trafficking.
Activators of Beclin 1/Vps34 complex stimulate Beclin 1-dependent lipid kinase activity of Vps34. Vps34 kinase activity upregulates the phosphatidylinositol 3-phosphates (PI3P) at the phagophore. Activators of Beclin 1/Vps34 complex increase PI3P production in a cell.
The mechanistic target of rapamycin, also known as mammalian target of rapamycin (mTOR), is a protein encoded in humans by the MTOR gene. mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. mTOR belongs to the phosphatidylinositol 3-kinase-related kinase protein family and it is the catalytic subunit of two structurally distinct complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 is composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8 (MLST8) and the non-core components PRAS40 and DEPTOR. Upon inhibition, mTOR induces autophagy. In particular, mTORC1 inhibition. e.g. by amino acid starvation or pharmacological inhibition, leads to de-repression of ULK kinase activity. The active ULK directly phosphorylates Beclin-1 and activates Beclin 1-Vps34 complex.
An exemplary synthetic peptide capable of activating Beclin 1-Vps34 complex and called Tat-Beclin 1 peptide has been recently disclosed by Shoji-Kawata et al. (Nature 2013). Tat-Beclin 1 (also known as Atg6 Activator I, Beclin 1-GAPR-1 Interaction Blocker I, Vps30 Activator I, Autophagy Inducer IV) is a cell-permeable peptide that is composed of essential HIV-1 virulence factor Nef-binding sequence derived from human Atg6/Beclin 1 (aa 269-283) evolutionarily conserved domain (ECD) with substitutions at three non-species-conserved residues (H275E, S279D, and Q281E) for enhanced solubility and N-terminally fused to the membrane-permeant HIV-1 Tat protein transduction domain (PTD) sequence (aa 47-57) via a-Gly-Gly-linkage, to facilitate cellular delivery and Beclin 1 activation via competitive binding to its negative regulator “Golgi-associated plant pathogenesis-related protein-1” (GAPR-1/GLIPR2) on the Golgi surface. Tat-Beclin 1 peptide induces a complete cellular autophagy response. Tat-Beclin 1 peptide may promote the release of Beclin 1 from the Golgi, resulting in enhanced early autophagosome formation. Other unknown mechanisms may also contribute to the Beclin 1-Vps 34 complex activation and autophagy induction accomplished by Tat-Beclin 1.
Tat-Beclin 1 peptide treatment in multiple cell lines (e.g. HeLa, COS-7, MEFs, A549, HBEC30-KT, THP1, and HCC827 cells) leads to p62 degradation and LC3-II conversion.
Phosphatidylethanolamine (PE) conjugation of mammalian LC3 results in a non-soluble form of LC3 (LC3-II) that stably associates with the autophagosomal membrane. Lipidated LC3 (LC3-II), but not unlipidated LC3 (LC3-I), binds to autophagosomes and LC3 lipidation correlates with autophagosome formation. When autophagy is induced, western blot analysis reveals that LC3-II protein levels are increased.
p62 protein is selectively degraded by the autophagy machinery and its protein levels reflects the amount of autophagic flux (i.e. a complete autophagy response). When autophagy is induced, western blot analysis reveals that p62 protein levels are decreased.
Also a retro-inverso Tat-Beclin 1 peptide has been disclosed (Shoji-Kawata et al., 2013), which is capable of activating Beclin 1/Vps34 complex: the retro-inverso Tat-Beclin 1 peptide (also known as Atg6 Activator II, Beclin-1-GAPR-1 Interaction Blocker II, Vps30 Activator II), consists in the all-D-amino acid retro-inverso sequence of Tat-Beclin 1.
Administration to mice of any of the two peptides leads to increase of autophagosomes in peripheral tissues (skeletal muscles and cardial muscles, pancreas, at 20 mg/kg i.p.) and increase of autophagosomes in central nervous system of neonatal mice (15 mg/kg, 1/die for 2 weeks). Daily treatment with Tat-Beclin 1 peptides for 2 weeks in adult and neonatal mice is well-tolerated. Efficient reduction of infections in mice infected with CHKN (muscle, skin, joints) or WNV (CNS) consequent to administration of Tat-Beclin 1 peptide is also shown by Shoji-Kawata et al. (Nature, 2013). No further therapeutic effects, nor activity, of a Tat-Beclin 1 peptide or derivatives thereof have ever been shown in skeletal tissue.
Beclin 1 peptide analogues, fragments or derivatives thereof, such as Tat-Beclin 1 peptide, are disclosed in WO2013119377 and WO2014149440 incorporated by reference. The use of said peptides, analogues, fragments or derivatives thereof for the treatment of bone-related disorders has never been disclosed nor suggested.
WO2011106684 discloses Beclin-1 derivative peptides of sequence comprising all or a subsequence of Beclin 1, fused to the protein transduction domain of an HIV Tat protein. WO2011106684 generally refers to the use of autophagy modulators for treating diseases with dysregulated autophagy. Among others, lysosomal storage disorders are mentioned, however a direct correlation between the use of an autophagy inducer or of Beclin-1 derivative peptides and the treatment of lysosomal storage disorders is not disclosed.
WO201128941 claims methods of treating lysosomal storage disease through inhibition of autophagy.
Shapiro et al (Autophagy, 2014) disclose that patients and mouse models of LSDs display a higher number of autophagosomes, most likely resulting from a defective lysosome-autophagosome fusion. Furthermore, it discloses that treatment of rats with the autophagy activator rapamycin impairs longitudinal growth.
Alvarez-Garcia et al. (Pediatr Nephrol, 2007) disclose that rapamycin impairs longitudinal growth in young rats, causing marked alterations in the growth plate, and that rapamycin disrupts angiogenesis and decreases proliferation and hypertrophy of growth cartilage chondrocytes. In humans, Gonzalez et al (Pediatr Nephrol, 2010) disclose lower growth rate in a small series of kidney transplanted children treated with rapamycin in comparison with a control group not treated with rapamycin.
Settembre et al. (Autophagy, 2009) disclose that autophagy is important for chondrocyte metabolism during endochondral ossification, and also hypothesize that its impairment may contribute to the development of skeletal abnormalities, such as those observed in MSD. However, they do not provide any evidence or suggestion that induction of autophagy and in particular that activation of the Beclin-1/Vps34 complex could be effective in the treatment of bone disorders.
The inventors have unexpectedly shown that alterations of the autophagic cellular function primary lead to bone growth disorders.
Surprisingly, molecules that are capable of activating Beclin-1/Vps34 complex, which is involved in initiation of the autophagic pathway and in the regulation of endocytic vesicles trafficking, efficiently prevent and/or treat bone growth pathologies.
The present invention provides an activator of beclin 1-Vps 34 complex for use in the treatment and/or prevention of a bone growth disorder wherein said activator is selected from the group consisting of:
Preferably the activator increases phosphatidylinositol 3-phosphates (PI3P) production in a cell.
Preferably the functional fragment comprises residues 270-278 of SEQ ID No. 43. In the present invention the functional derivatives may be functional derivatives of SEQ ID No. 43 or of a functional fragment thereof. For instance the functional derivatives may be the derivative of a functional fragment comprising residues 270-278 of SEQ ID No. 43. Functional derivatives are defined below.
Yet preferably the functional fragment is flanked by no more than twelve naturally-flanking Beclin 1 residues. This means that on each sides (at N and C terminal of residues 270-278 of SEQ ID NO:43) a maximum of 12 amino acids can be present. Such amino acid may be the same amino acid present in Beclin 1 in these positions (i.e. “naturally-flanking” Beclin 1 residues).
Preferably the functional derivative comprises SEQ ID NO: 43 or a functional fragment thereof and wherein said functional derivative comprises from 1 to 6 amino acid residue substitution(s) and/or a heterologous moiety.
Preferably the heterologous moiety consists of SEQ ID No. 44 or SEQ ID No. 45.
Preferably the polypeptide or the functional fragment thereof or the functional derivative thereof is partially or fully cyclized.
In a preferred embodiment the polypeptide is a retro-inverso polypeptide.
Still preferably the polypeptide comprises a sequence selected from the group consisting of: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 12 to SEQ ID No. 38 or a functional fragment thereof or a functional derivative thereof.
In a preferred embodiment the activator is a polynucleotide encoding for the polypeptide as defined in any of claims 3 to 9, preferably the polynucleotide comprises SEQ ID NO: 7.
In a preferred embodiment the activator is a vector comprising the polynucleotide as defined above, preferably said vector is a viral vector.
Preferably the activator further comprises a polynucleotide coding for the wild-type form of the protein whose mutated form is responsible for the bone growth disorder or a vector comprising said polynucleotide or further comprising the wild-type form of a protein whose mutated form is responsible for the bone growth disorder.
Preferably the protein whose mutated form is responsible for the bone growth disorder is selected from the group consisting of: FGFR3, FGFR1, FGFR2, FGFR4, β-glucocerebrosidase, α-mannosidase, α-fucosidase, α-neuraminidase, Cathepsin-A, UDP-N-acetylglucosamine, N-acetylglucosamine-1-phosphotransferase, Sulfatase modifying factor 1, Cathepsin K, α-L-iduronidase, Iduronate-2-sulfatase, Heparan N-sulfatase, α-N-acetyl glucosaminidase, Acetyl-CoA: α-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-6-sulfatase, β-D-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase and Hyaluronidase.
In a preferred embodiment the inhibitor of mTORC1 is selected from the group consisting of: Rapamycin, KU0063794, WYE354, Deforolimus, TORN 1, TORN 2, Temsirolimus, Everolimus, sirolimus, NVP-BEZ235 and PI103.
In a yet preferred embodiment the bone growth disorder is selected from the group consisting of: achondroplasia, hypochondroplasia, spondyloepiphyseal dysplasia, a lysosomal storage disorder, preferably a mucopolysaccharidosis (MPS).
Preferably the lysosomal storage disorder is selected from the group consisting of: MPS I, MPS II, MPS IV, MPS VI, MPS VII, MPS IX, Gaucher disease type 3, Gaucher disease type 1, multiple sulfatase deficiency, mucolipidosis type II, mucolipidosis type III, galactosidosis, alpha-mannosidosis, beta-mannosidosis, fucosidosis, pycnodysostosis.
Still preferably the bone growth disorder is selected from the group consisting of: achondroplasia, MPS VI and MPS VII.
The present invention also provides a pharmaceutical composition for use in the treatment and/or prevention of a bone growth disorder comprising the activator as defined above and pharmaceutically acceptable carriers.
Preferably the pharmaceutical composition further comprises a polynucleotide coding for the wild-type form of the protein whose mutated form is responsible for the bone growth disorder or a vector comprising said polynucleotide or further comprising the wild-type form of a protein whose mutated form is responsible for the bone growth disorder.
Preferably the pharmaceutical composition further comprises a therapeutic agent, preferably the therapeutic agent is selected from: enzyme replacement therapy, growth hormone, BMN111.
The present invention also provides a method for the treatment and/or prevention of a bone growth disorder in a subject in need thereof comprising administering an effective amount of the activator as defined above or the pharmaceutical composition as defined above.
The present invention also provides a vector for use in the treatment and/or prevention of a bone growth disorder said vector comprising a polynucleotide coding for an activator of beclin 1-Vps 34 complex, wherein said activator of beclin 1-Vps 34 complex is a polypeptide comprising a Beclin 1 peptide consisting of SEQ ID No. 43 or a functional fragment thereof or a functional derivative thereof, preferably, the functional fragment comprises residues 270-278 of SEQ ID No. 43, preferably the functional derivative comprises SEQ ID NO: 43 or a functional fragment thereof and said functional derivative comprises from 1 to 6 amino acid residue substitution(s) and/or a heterologous moiety.
Preferably the polynucleotide encodes a peptide consisting of a sequence selected from the group consisting of: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 12 to SEQ ID No. 38 or a functional fragment thereof or a functional derivative thereof.
Still preferably the polynucleotide comprises SEQ ID No. 3, preferably it is a viral vector, preferably an adeno-associated vector (AAV).
Yet preferably the vector further comprises a polynucleotide coding for the wild-type form of the protein whose mutated form is responsible for a bone growth disorder.
According to a preferred embodiment, the activator of the invention is a peptide comprising the sequence YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID NO: 1, herein Tat-Beclin 1 peptide), or a functional fragment or a functional derivative thereof.
According to a preferred embodiment, the activator of the invention is a peptide comprising the sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID NO: 2, herein retro-inverso Tat-Beclin 1 or (D)-Tat-Beclin 1) or a functional fragment or a functional derivative thereof.
In the present invention functional fragments of SEQ ID No. 43 and functional derivatives maintain the biological activity of increasing phosphatidylinositol-3-phosphates production, which can be easily measured by methods known in the art.
The present invention is directed to a molecule capable of activating Beclin 1/Vps34 complex in a cell for use in the treatment and/or prevention of a bone growth disorder; more preferably, said cell is a chondrocyte; most preferably said cell is a mammalian cell.
An activator of Beclin 1/Vps34 is a molecule that favors vps34 PI3K Beclin 1-dependent activity. Activation of Beclin 1/Vps34 complex directly leads to the increase of PI3P levels. In other words, activators of Beclin 1/Vps34 complex stimulate Beclin 1-dependent lipid kinase activity of Vps34. Vps34 kinase activity upregulates the phosphatidylinositol 3-phosphates (PI3P) at the phagophore. Activators of Beclin 1/Vps34 complex increase PI3P production in a cell.
Activation of Beclin 1/Vps34 complex can thus be assessed by any assay for measuring the levels of PI3P at the phagophore. An exemplary assay is the membrane-associated PI3 Kinase (PI3K) assay in situ, as described herein. FYVE is a domain that binds with great specificity to PI3P. 2×FYVE-EGFP transfected in a cell localizes to early endosomes in a PI3K activity-dependent fashion (Pattini et al, 2001). In cells transfected with GFP-2⋅FYVE and then treated with a potential activator of Beclin 1/Vps34 complex, EGFP puncta increase compared to control cells (vehicle treated). Further methods include the analysis of PI3K activity in Beclin 1 immunoprecipitates, using commercial PI3K ELISA kits, according to the manufacturer's instructions.
An inhibitor of mTORC1 is a molecule capable of prevent either phosphorylation of proteins substrates or autophosphorylation of mTOR. In particular, an activator of Beclin 1/Vps34 complex, which is an inhibitor of mTORC1, according to a preferred embodiment of the invention, is a molecule capable of reducing ULK1 phosphorylation by mTORC1.
ULK1 phosphorylation reduction can be assessed for example as described herein by measuring the relative levels of Phospo-ULK1 proteins.
A small molecule is a low molecular weight (<900 daltons) organic compound, with a size on the order of 109 m. A small molecule binds to a specific biological target—such as a specific protein or nucleic acid—and acts as an effector, altering the activity or function of the target.
Preferred inhibitors of mTORC1 comprise: Rapamycin (CAS No. 53123-88-9), KU0063794 (CAS No. 938440-64-3), WYE354 (CAS No. 1062169-56-5), Deforolimus (CAS No. 572924-54-0), TORN 1 (CAS No. 1222998-36-8), TORN 2 (CAS No. 1223001-51-1), Temsirolimus (CAS No. 162635-04-3), Everolimus (CAS No. 159351-69-6), sirolimus (CAS No. 53123-88-9), NVP-BEZ235 (CAS No. 915019-65-7), PI103 (CAS No. 371935-74-9).
BH3 mimetics are small molecules capable of mimicking BH3-only proteins of the BCL-2 family, i.e. having only the BCL-2 homology domain BH3.
Bcl-2 homology (BH) domains: BH3 domain in Beclin 1 is similar to that required for the binding of proapoptotic proteins to antiapoptotic Bcl-2 homologs. Typically, a BH3 domain is defined as a four-turn amphipatic α-helix, bearing the sequence motif: Hy-X-X-X-Hy-K/R-X-X-Sm-D/E-X-Hy, in which Hy are hydrophobic residues and Sm represents small residues, typically glycine. Proapoptotic Bcl-2 proteins are grouped into two categories: (1) the multidomain proapoptotic proteins that contain three BH domains, BH4, BH3 and BH1; and (2) the BH3-only proapoptotic proteins that contain only the BH3 domain. In both these groups, the BH3 domain is required for interaction with antiapoptotic Bcl-2 proteins. The BH3-only proteins are thus a subset of the Bcl-2 family of proteins, containing only a single BH3-domain. The BH3-only family members are Bim, Bid, BAD and others. Various apoptotic stimuli induce expression and/or activation of specific BH3-only family members, which translocate to the mitochondria and initiate Bax/Bak-dependent apoptosis. BH3-mimetics promote dissociation of Beclin-1 from BclXL thus making Beclin-1 able to enter into the initiating complex comprising Vps34 and Vps15. Preferred BH3 mimetics for use according to the present invention comprise: ABT-737, ABT-263/navitoclax, Obatoclax, Gossypol, AT-101, Apogossypol, Apogossypolone/ApoG2, BI-97C1/sabutoclax, TW37, S1, 072RB, SAHB-A, BIMS2A, Mc1-1 SAHB (Billard, 2013).
In the present invention a Beclin 1 peptide refers to accession number NP_003757 (SEQ ID No. 45)
or a peptide encoded by an ortholog gene thereof.
In the present invention a Beclin 1 peptide fragment is a peptide comprising a subsequence of a Beclin 1 peptide; a Beclin 1 peptide fragment is thus a peptide shorter than the Beclin 1 peptide whose sequence is reported above; preferably said fragment or subsequence comprises residues 270-278 of a Beclin 1 peptide, more preferably it comprises residues 269-283 of a Beclin 1 peptide. Preferably said Beclin 1 fragment comprises at least 3 amino acid residues, preferably at least 5, at least 6, at least 8, at least 10, at least 15 or at least 20 amino acid residues. Preferably, said Beclin 1 peptide fragment has at least 65%, at least 70%, at least 80%, at least 90%, at least 95% identity with the Beclin 1 peptide. Said Beclin 1 peptide fragment maintains the biological activity of Beclin 1, i.e. activation of the Beclin 1/Vps34 complex, so that said fragment may treat or prevent a bone growth disorder.
In the present invention a Beclin 1 peptide derivative is a peptide comprising a Beclin 1 peptide or a Beclin 1 peptide fragment or the retro-inverso peptide thereof, and comprising alternative structures and/or formulations of said Beclin 1 peptide or of said Beclin 1 peptide fragment or of said retro-inverso peptide thereof.
For instance, said Beclin 1 derivative peptide may comprise at least one heterologous moiety (i.e. a moiety deriving from a different species), and/or may be chemically modified. The derivative maintains the biological activity of Beclin 1, i.e. activation of the Beclin 1/Vps 34 complex, so that said derivative may treat or prevent a bone growth disorder. In an exemplary non-limiting embodiment, a Beclin 1 peptide derivative is a peptide comprising residues 270-278 of a Beclin 1 peptide, optionally flanked by no more than twelve naturally-flanking Beclin 1 residues, wherein up to six residues may be substituted, and linked to a heterologous moiety. According to an exemplary non-limiting embodiment, a peptide derivative is a peptide comprising the Beclin 1 peptide or a fragment thereof or a retro-inverso peptide thereof and having amino acid residue substitution(s). Preferably said derivative comprises from 1 to 6 amino acid residue substitution(s).
Retro-inverso peptides are linear peptides whose amino acid sequence is reversed and the α-center chirality of the amino acid subunits is inverted as well. Usually, these types of peptides are designed by including D-amino acids in the reverse sequence to help maintain side chain topology similar to that of the original L-amino acid peptide and make them more resistant to proteolytic degradation. Other reported synonyms for these peptides in the scientific literature are: Retro-Inverso Peptides, All-D-Retro Peptides, Retro-Enantio Peptides, Retro-Inverso Analogs, Retro-Inverso Analogues, Retro-Inverso Derivatives, and Retro-Inverso Isomers. D-amino acids represent conformational mirror images of natural L-amino acids occurring in natural proteins present in biological systems. Peptides that contain D-amino acids have advantages over peptides that just contain L-amino acids. In general, these types of peptides are less susceptible to proteolytic degradation and have a longer effective time when used as pharmaceuticals. Furthermore, the insertion of D-amino acids in selected sequence regions as sequence blocks containing only D-amino acids or in-between L-amino acids allows the design of peptide based drugs that are bioactive and possess increased bioavailability in addition to being resistant to proteolysis. Furthermore, if properly designed, retro-inverso peptides can have binding characteristics similar to L-peptides. Retro-inverso-peptides are attractive alternatives to L-peptides used as pharmaceuticals. These type of peptides have been reported to elicit lower immunogenic responses compared to L-peptides. In the present invention a retro-inverso sequence is thus a reversed sequence wherein the α-center chirality of the amino acid subunits is inverted as well. Preferably, the retro-inverso peptide comprises all D-amino acids. As an example: the retro-inverso peptide of a peptide of sequence VFNATFHIWHSGQFG (SEQ ID No. 13) would be a peptide of sequence GFQGSHWIHFTANFV (SEQ ID No. 46). The availability of modern chemical synthesis methods allows the routine synthesis of these types of peptides.
Preferably, the molecule of the invention is for use in the treatment and/or prevention of a bone growth disorders. Exemplary bone growth disorders include achondroplasia, hypochondroplasia, MPS I, MPS II, MPS IV, MPS VI, MPS VII, MPS IX, Gaucher disease type 3, Gaucher disease type 1, a glycoproteinoses, pycnodysostosis. Further bone growth disorders include bone disorders with collagen involvement such as the group of spondyloepiphyseal dysplasias.
Beclin 1/Vps34 complex is a protein complex comprising Beclin 1 protein (NP_003757) and Vps34 protein (NP_001294949; NP_002638). The activation of said complex is capable of inducing autophagic response in a cell; as an example the activation of said complex can induce the first step of autophagosome formation, the nucleation of the phagophore at the endoplasmic reticulum (autophagic vesicle nucleation). Further components of the active Beclin-1/Vps34 complex include Vps15 protein (NP_055417). Optionally the active Beclin-1/Vps34 complex includes Atg14L (NP_055739); optionally the active Beclin-1/Vps34 complex includes UVRAG protein (NP_003360); optionally the active Beclin-1/Vps34 complex includes Ambral protein (NP_060219). Preferably, the active Beclin 1/Vps34 complex does not include Rubicon protein (NP_001139114), which has been shown to negatively regulate the Beclin 1/Vps34 complex.
Preferably, the molecule of the invention for use in the treatment of a bone growth disorder, capable of activating a Beclin 1/Vps34 complex, induces autophagy and/or promotes endocytic trafficking. Therefore, preferably, a molecule capable of activating Beclin 1/Vps34 complex in a cell is a molecule capable of inducing autophagy in a cell, more preferably a molecule capable of inducing formation of autophagosomes and of autophagosomes-lysosome fusion in a cell.
In order to assess autophagic cellular response, autophagosome biogenesis (WIPI2 and Atg16 positive dots), maturation (LC3-LAMP1 positive vesicles) and substrate degradation (long lived protein and p62 degradation) rates can be measured.
In a cell or tissue, activation of Beclin 1-Vps 34 complex can be detected directly, indirectly or inferentially by conventional assays, such as disclosed and/or exemplified herein. Activation of Beclin 1/Vps34 complex in a cell can be assessed by several methods known on the art. In particular, the activation of Beclin 1/Vps34 can be assessed and measured by measuring the PI3P production in in a cell, in a tissue and/or in the growth plates from treated and untreated subjects. Further methods include the quantification by western blot and immunofluorescence analyses of the levels of p62, LAMP1 and LC3II proteins in a cell, in a tissue and/or in the growth plates from treated and untreated subjects.
An activator according to the invention for use in the treatment and/or prevention of a bone growth disorder can thus be identified by quantification by western blot and/or immunofluorescence analyses of the levels of p62, LAMP1 and LC3II proteins.
In order to quantify the fraction of lysosome and autophagosome vesicles at different stages of maturation transmission electron microscopy on growth plate and cortical bone sections of treated and untreated subjects can be performed.
mTORC1 activity can be assessed by measuring the relative levels of phospho-p70 S6K and of Phospo-ULK1 proteins in growth plate and bone extracts. Also, the intracellular localization of TFEB and of TFE3 (nuclear vs cytosolic) by immunohistochemistry can be monitored, as well as the expression levels of autophagy and lysosomal genes by qPCR. Inhibition of mTORC leads to activation of Beclin 1/Vps34 complex and consequently to induction of cellular autophagy/endocytic trafficking. Inhibition of mTORC can thus be measured by the assays herein described aimed at measuring activation of Beclin 1/Vps34 complex.
Preferably, the molecule of the invention for use in the treatment of a bone growth disorder is selected from the group comprising: a Beclin 1 peptide fragment, a Beclin 1 derivative peptide, an mTORC1 inhibitor or a BH3 mimetic.
According to a preferred embodiment, the molecule of the invention for use in the treatment of a bone growth disorder is a Beclin 1 peptide fragment comprising residues 270-278 of Beclin 1 protein sequence or a fragment comprising residues 269-283 of Beclin 1 protein sequence, or retro-inverso sequence thereof.
According to a preferred embodiment, the molecule of the invention for use in the treatment of a bone growth disorder is a Beclin 1 derivative peptide; more preferably said Beclin 1 derivative peptide comprises: (a) residues 269-283 of Beclin 1 protein sequence immediately flanked on each terminus by no more than twelve naturally-flanking Beclin 1 residues, wherein up to six of said residues 269-283 may be substituted, and (b) a first heterologous moiety.
According to preferred embodiments, the molecule of the invention for use in the treatment of a bone growth disorder can consist in a Beclin 1 derivative peptide, said Beclin 1 derivative peptide comprising: (a) residues 269-283 of Beclin 1 protein sequence (VFNATFHIWHSGQFG; SEQ ID NO:13) immediately flanked on each terminus by no more than twelve naturally-flanking Beclin 1 residues, wherein up to six of said residues 269-283 may be substituted, and (b) a first heterologous moiety, such as wherein:
the peptide is N-terminally flanked with T-N and C-terminally flanked by T;
the peptide comprises at least one of F270, F274 and W277;
the peptide comprises at least one substitution, particularly of H275E, S279D or Q281E;
the peptide is N-terminally joined to the first moiety, and C-terminally joined to a second heterologous moiety;
the peptide is joined to the first moiety through a linker or spacer; preferably the linker or spacer is a a diglycine linker. the first moiety comprises a transduction domain, including: protein-derived (e.g. Tat (SEQ ID NO: 44), smac (Accession number GenBank: AAF87716.1), pen (ALC39141.1), pVEC, bPrPp (ALS90899.1), PIs1 (A1RQH3.1), VP22 (ANR01123.1), M918 (EQB90450.1), pep-3 (AAA34852.1)), chimeric (e.g. TP (CAE48349.1), TP10 (CAI48908.1), MPGA (XP 637125.1)), and synthetic (e.g. MAP (CAJ99007.1), Pep-1 (AAQ01688.1), oligo-Arg cell-penetrating peptides;
the first moiety comprises a homing peptide, such as RGD-4C, NGR (Q9N0E3.1), CREKA, LyP-1 (XP_009259791.1), F3 (ABA26022.1), SMS (AAA97285.1), IF7 (NP_035129.1) or H2009.1 (AIG45257.1);
the first moiety comprises a stabilizing agent, such as a PEG, oligo-N-methoxyethyl glycine (NMEG), albumin, an albumin-binding protein, or an immunoglobulin Fc domain;
the peptide comprises one or more D-amino acids, L-P-homo amino acids, D-β-homo amino acids, or N-methylated amino acids;
the peptide is cyclized;
the peptide is acetylated, acylated, formylated, amidated, phosphorylated, sulfated or glycosylated;
the peptide comprises an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl or 2-furosyl group, and/or a C-terminal hydroxyl, amide, ester or thioester group;
the peptide comprises an affinity tag or detectable label; and/or the peptide is N-terminally joined to the first moiety, and C-terminally joined to a second heterologous moiety comprising a detectable label, such as a fluorescent label. Labels and tags are known in the art.
Particular embodiments include all combinations and sub-combinations of particular embodiments, such as wherein: the peptide is N-terminally flanked with T-N and C-terminally flanked by T, the first moiety is a tat protein transduction domain linked to the peptide through a diglicine linker; and the peptide is N-terminally flanked with T-N and C-terminally flanked by T, the first moiety is a tetrameric integrin a(v)P(6)-binding peptide known as H2009.1, linked to the peptide through a maleimide-PEG(3) linker.
In a preferred aspect of the invention the molecule is a Beclin 1 derivative peptide comprising: (a) Beclin 1 residues 269-283 (SEQ ID No. 13) immediately flanked on each terminus by no more than 12 (or 6, 3, 2, 1 or 0) naturally-flanking Beclin 1 residues, wherein up to six (or 3, 2, 1 or 0) of said residues 269-283 may be substituted, and (b) a first heterologous moiety. In some embodiments the peptide may be N-terminally flanked with TN and C-terminally flanked by T (TNVFNATFHIWHSGQFGT; SEQ ID NO:14). In some embodiments the peptide comprises at least one (or two or three) of substitutions: H275E, S279D and Q281E (e.g. VFNATFEIWHDGEFG; SEQ ID NO:15).
In other embodiments the peptide comprises at least one (or two or three) of F270, F274 and W277.
Peptides activity according to preferred embodiments of the invention are also tolerant to backbone modification and replacement, side-chain modifications, and N- and C-terminal modifications, all conventional in the art of peptide chemistry.
Chemical modifications of the peptides bonds may be used to provide increased metabolic stability against enzyme-mediated hydrolysis; for example, peptide bond replacements (peptide surrogates), such as trifluoroethylamines, can provide metabolically more stable and biologically active peptidomimetics.
Modifications to constrain the peptides backbone include, for example, cyclic peptides/peptidomimetics which can exhibit enhanced metabolic stability against exopeptidases due to protected C- and N-terminal ends. Suitable techniques for cyclization include Cys-Cys disulfide bridges, peptide macrolactam, peptide thioether, parallel and anti-parallel cyclic dimers, etc.
Other suitable modifications include acetylation, acylation (e.g. lipopeptides), formylation, amidation, phosphorylation (on Ser, Thr and/or Tyr), etc. which can be used to improve peptide bioavailability and/or activity, glycosylation, sulfonation, incorporation of chelators (e.g. DOTA, DPTA), etc. PEGylation can be used to increase peptide solubility, bioavailability, in vivo stability and/or decrease immunogenicity, and includes a variety of different PEGs: HiPEG, branched and forked PEGs, releasable PEGs; heterobifunctional PEG (with endgroup N-Hydroxysuccinimide (NHS) esters, maleimide, vinyl sulfone, pyridyl disulfide, amines, and carboxylic acids), etc.
Suitable terminal modifications include N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl and 2-furosyl, and C-terminal hydroxyl, amide, ester and thioester groups, which can make the peptide more closely mimic the charge state in the native protein, and/or make it more stable to degradation from exopeptidases.
According to preferred embodiments, the peptides may also contain atypical or unnatural amino acids, including D-amino acids, L-P-homo amino acids, {umlaut over (ν)}-β-homo amino acids, N-methylated amino acids, etc.
In a particular embodiment, the peptide is N-terminally joined to a first moiety, heterologous to (not naturally flanking) the Beclin 1 peptide, typically one that promotes therapeutic stability or delivery, and C-terminally joined to a second moiety, preferably also heterologous to the Beclin 1 peptide. A wide variety of such moieties may be employed, such as affinity tags, transduction domains, homing or targeting moieties, labels, or other functional groups, such as to improve bioavailability and/or activity, and/or provide additional properties.
One useful class of such moieties include transduction domains which facilitate cellular penetrance or uptake, such as protein-derived (e.g. tat, smac, pen, pVEC, bPrPp, PIs1, VP22, M918, pep-3); chimeric (e.g. TP, TP10, MPGA) or synthetic (e.g. MAP, Pep-1, Oligo Arg) cell-penetrating peptides; see, e.g. “Peptides as Drugs: Discovery and Development”, Ed. Bernd Groner, 2009 WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim, esp. Chap 7: “The Internalization Mechanisms and Bioactivity of the Cell-Penetrating Peptides”, Mats Hansen, Elo Eriste, and Ulo Langel, pp. 125-144.
Another class are homing biomolecules, such as RGD-4C, NGR, CREKA, LyP-1, F3, SMS (SMSIARL, SEQ ID No. 47), IF7, and H2009.1 (Li et al. Bioorg Med Chem. 2011 Sep. 15; 19(18):5480-9), particularly cancer cell homing or targeting biomolecules, wherein suitable examples are known in the art, e.g. Homing peptides as targeted delivery vehicles Pirjo Laakkonen and Kirsi Vuorinen, Integr. Biol., 2010, 2, 326-337; Mapping of Vascular ZIP Codes by Phage Display, Teesalu T, Sugahara K N, Ruoslahti E., Methods Enzymol. 2012; 503:35-56.
Other useful classes of such moieties include stabilizing agents, such as PEG, oligo-N-methoxyethylglycine (NMEG), albumin, an albumin-binding protein, or an immunoglobulin Fc domain; affinity tags, such as immuno-tags, biotin, lectins, chelators, etc.; labels, such as optical tags (e.g. Au particles, nanodots), chelated lanthanides, fluorescent dyes (e.g. FITC, FAM, rhodamines), FRET acceptor/donors, etc.
The moieties, tags and functional groups may be coupled to the peptide through linkers or spacers known in the art, such as polyglycine, c-aminocaproic, etc.
The peptide can also be presented as latent or activatable forms, such as a prodrug, wherein the active peptide is metabolically liberated; for example, release of the linear peptide from cyclic prodrugs prepared with an acyloxyalkoxy promoiety (prodrug 1) or a 3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-dimethyl propionic acid promoiety (prodrug 2) of the peptide).
According to a preferred embodiment, said peptide comprises one or more D-amino acids, L-β-homo amino acids, O-β-homo amino acids, or N-methylated amino acids.
According to a preferred embodiment, said compound comprises an affinity tag or detectable label.
According to a preferred embodiment, said peptide is N-terminally joined to the first moiety, and C-terminally joined to a second heterologous moiety comprising a fluorescent label.
According to a preferred embodiment, said peptide is N-terminally flanked with T-N and C-terminally flanked by T, the first moiety is a tat protein transduction domain linked to the peptide through a diglycine linker.
According to a preferred embodiment, said peptide is N-terminally flanked with T-N and C-terminally flanked by T, the first moiety is a tetrameric integrin a(v)P(6)-binding peptide known as H2009.1, linked to the peptide through a maleimide-PEG(3) linker.
In a further aspect of the invention, the molecule capable of activating Beclin-1/Vps34 complex for use in the treatment and/or prevention of a bone growth disorder is a Beclin 1 derivative peptide comprising Beclin 1 residues 270-278 (FNATFHIWH; SEQ ID NO: 16), or the D-retro-inverso sequence thereof, immediately N- and C-terminally flanked by moieties R1 and R2, respectively, wherein up to six of said residues may be substituted, R1 and R2 do not naturally flank the Beclin 1 residues, and F270 and F274 are optionally substituted and optionally linked.
In particular embodiments of the invention said peptide's sequence is unsubstituted or up to six of said residues may be substituted, and the two F residues are F1 and F2 and are optionally substituted and optionally linked, or said compound has D-retro-inverso sequence of said peptide; optionally wherein:
The invention includes all combinations of the recited particular embodiments above, as if each combination had been laboriously separately recited.
Peptides and compound activity are tolerant to a variety of additional moieties, flanking residues, and substitutions within the defined boundaries. Peptide and compound activity are also tolerant to backbone modification and replacement, side-chain modifications, and N- and C-terminal modifications, all conventional in the art of peptide chemistry.
Chemical modifications of the peptides bonds may be used to provide increased metabolic stability against enzyme-mediated hydrolysis; for example, peptide bond replacements (peptide surrogates), such as trifluoroethylamines, can provide metabolically more stable and biologically active peptidomimetics.
Modifications to constrain the peptides backbone include, for example, cyclic peptides/peptidomimetics which can exhibit enhanced metabolic stability against exopeptidases due to protected C- and N-terminal ends. Suitable techniques for cyclization include Cys-Cys disulfide bridges, peptide macrolactam, peptide thioether, parallel and anti-parallel cyclic dimers, etc. ; see, e.g. PMID 22230563 (stapled peptides), PMID 23064223 (use of click variants for peptide cyclization), PMID 23133740 (optimizing PK properties of cyclic peptides: effects of side chain substitutions), PMID: 22737969 (identification of key backbone motifs for intestinal permeability, PMID 12646037 (cyclization by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII), and by replacing Asn with this lipoamino acid).
In particular embodiments F270 and F274 are substituted and linked, such as wherein the side chains of F270 and F274 replaced by a linker. For example, these residues may be substituted with homocysteines connected through a disulfide bridge to generate a ring and tail cyclic peptide. In addition, the side chains of these residues can be substituted and cross-linked to form a linker, such as —CH2)nONHCOX(CH2)m-, wherein X is C3/4, NH or O, and m and n are integers 1-4, forming a lactam peptide; —CH2OCH2CHCHCH2OCH2-, forming an ether peptide; —(CH2)nCHCH(CH2)m-, forming a stapled peptide. The linkers may incorporate additional atoms, heteroatoms, or other functionalities, and are typically generated from reactive side chain at F270 and F274. The crosslinkable moieties may include additional α-carbon substititions, such as optionally substituted, optionally hetero-lower alkyl, particularly optionally substituted, optionally hetero-methyl, ethyl, propyl and butyl. Suitable modifications include acetylation, acylation, formylation, amidation, phosphorylation (on Ser, Thr and/or Tyr), etc. which can be used to improve peptide bioavailability and/or activity, glycosylation, sulfonation, incorporation of chelators (e.g. DOTA, DPT A), etc. PEGylation can be used to increase peptide solubility, bioavailability, in vivo stability and/or decrease immunogenicity, and includes a variety of different PEGs: HiPEG, branched and forked PEGs, releasable PEGs; heterobifunctional PEG (with endgroup N-Hydroxysuccinimide (NHS) esters, maleimide, vinyl sulfone, pyridyl disulfide, amines, and carboxylic acids), etc.
Suitable terminal modifications include N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl and 2-furosyl, and C-terminal hydroxyl, amide, ester and thioester groups, which can make the peptide more closely mimic the charge state in the native protein, and/or make it more stable to degradation from exopeptidases. [038] The peptides may also contain atypical or unnatural amino acids, including D-amino acids, L-homo amino acids, O-β-homo amino acids, N-methylated amino acids, etc.
A wide variety of flanking moieties R1 and/or R2 may be employed, such as affinity tags, transduction domains, homing or targeting moieties, labels, or other functional groups, such as to improve bioavailability and/or activity, and/or provide additional properties.
One useful class of such moieties include transduction domains which facilitate cellular penetrance or uptake, such as protein-derived (e.g. tat, smac, pen, pVEC, bPrPp, PIs1, VP22, M918, pep-3); chimeric (e.g. TP, TP10, MPOA) or synthetic (e.g. MAP, Pep-1, Oligo Arg) cell-penetrating peptides; see, e.g. “Peptides as Drugs: Discovery and Development”, Ed. Bernd Groner, 2009 WILEY-VCH Verlag GmbH & Co, KGaA, Weinheim, esp. Chap 7: “The Internalization Mechanisms and Bioactivity of the Cell-Penetrating Peptides”, Mats Hansen, Elo Eriste, and Ulo Langel, pp. 125-144.
Another class are homing biomolecules, such as RGD-4C, NGR, CREKA, LyP-1, F3, SMS (SMSIARL), IF7, and H2009.1 (Li et al. Bioorg Med Chem. 2011 Sep. 15; 19(18):5480-9), particularly cancer cell homing or targeting biomolecules, wherein suitable examples are known in the art, e.g. Homing peptides as targeted delivery vehicles, Pirjo Laakkonen and Kirsi Vuorinen, Integr. Biol., 2010, 2, 326-337; Mapping of Vascular ZIP Codes by Phage Display, Teesalu T, Sugahara K N, Ruoslahti E., Methods Enzymol. 2012; 503:35-56.
Other useful classes of such moieties include stabilizing agents, such as PEG, oligo-N-methoxyethylglycine (NMEG), albumin, an albumin-binding protein, or an immunoglobulin Fc domain; affinity tags, such as immuno-tags, biotin, lectins, chelators, etc.; labels, such as optical tags (e.g. Au particles, nanodots), chelated lanthanides, fluorescent dyes (e.g. FITC, FAM, rhodamines), FRET acceptor/donors, etc.
The moieties, tags and functional groups may be coupled to the peptide through linkers or spacers known in the art, such as polyglycine, c-aminocaproic, etc.
The compound and/or peptide can also be presented as latent or activatable forms, such as a prodrug, wherein the active peptide is metabolically liberated; for example, release of the linear peptide from cyclic prodrugs prepared with an acyloxyalkoxy promoiety (prodrug 1) or a 3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-dimethyl propionic acid promoiety (prodrug 2). of the compound).
According to preferred embodiments of the invention, the molecule for use in the treatment and/or prevention of a bone growth disorder is a Beclin 1 derivative peptide comprising a sequence, unsubstituted, selected from:
or the D-retro-inverso sequence of said peptides.
According to preferred embodiments of the invention, R1 of said compound comprises a transduction domain, a homing peptide, or a serum stabilizing agent.
According to preferred embodiments of the invention, R1 of said compound is a tat protein transduction domain linked to the peptide through a diglycine linker, particularly a diglycine-T-N linker.
According to preferred embodiments of the invention, R2 of said compound is carboxyl or comprises an affinity tag or detectable label, particularly a fluorescent label.
According to preferred embodiments of the invention F270 and F274 are substituted with cross-linkable moieties and/or linked, and each optionally comprises an additional α-carbon substitution selected from substituted, optionally hetero-lower alkyl, particularly optionally substituted, optionally hetero-methyl, ethyl, propyl and butyl; or F270 and F274 are substituted with homocysteines connected through a disulfide bridge to generate a ring and tail cyclic peptide.
According to preferred embodiments of the invention, the side chains of F270 and F274 are replaced by a linker:
—(CH2)nONHCOX(CH2)m-, wherein X is CH2, NH or O, and m and n are integers 1-4, forming a lactam peptide; —CH2OCH2CHCHCH2OCH2-, forming an ether peptide; or
—(CH2)nCHCH(CH2)m-, forming a stapled peptide.
According to preferred embodiments of the invention, 1 to 6 residues are alanine substituted; or the peptide comprises at least one of substitutions: H275E and S279D; or the peptide comprises one or more D-amino acids, E-β-homo amino acids, O-β-homo amino acids, or N-methylated amino acids; or the peptide comprises the D-retro-inverso sequence.
According to preferred embodiments of the invention, the peptide is acetylated, acylated, formylated, amidated, phosphorylated, sulfated or glycosylated.
According to preferred embodiments of the invention, the compound comprises an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl or 2-furosyl group, and/or a C-terminal hydroxyl, amide, ester or thioester group.
According to preferred embodiments of the invention, the peptide is cyclized.
Preferably, the molecule of the invention for use in the treatment of a bone growth disorder is a peptide of sequence comprising SEQ ID NO: 1 (Tat-Beclin 1), or derivatives thereof, or a polynucleotide encoding for said peptide of sequence comprising SEQ ID NO: 1, or for a derivative thereof.
According to a further preferred embodiment, the molecule of the invention for use in the treatment of a bone growth disorder is a peptide of sequence comprising SEQ ID NO: 2 (retro-inverso Tat-Beclin 1) or derivatives thereof, or a polynucleotide encoding for said peptide of sequence comprising SEQ ID NO: 2 or for a derivative thereof.
According to a preferred embodiment, the molecule of the invention is a vector comprising a polynucleotide encoding for a peptide of sequence SEQ ID NO: 1 or SEQ ID NO:2, or derivatives thereof.
According to a preferred embodiment, the molecule of the invention is a vector comprising an expression cassette, said expression cassette comprising a polynucleotide encoding for any of the Beclin 1 fragment peptides and Beclin 1 derivative peptides disclosed herein; preferably said polynucleotide encodes for a peptide of sequence SEQ ID NO: 1 or SEQ ID NO: 2, or derivatives thereof.
Preferably the polynucleotides encoding for the Beclin 1 fragment peptides and Beclin 1 derivative peptides of the vectors of the present invention are under the control of a regulatory sequence, such as a promoter. Regulatory sequences contemplated for use in said vectors, include but are not limited to, native gene promoters, a cytomegalovirus (CMV) promoter, a liver-specific promoter, and a cartilage-specific promoter. Exemplary liver-specific promoters include human thyroid hormone-globulin (TBG) promoter and alpha-antitrypsin (AAT) promoter. In some embodiments, the promoter is selected from the group consisting of cytomegalovirus (CMV) promoter of sequence SEQ ID No. 39, human thyroid hormone-globulin (TBG) promoter of sequence SEQ ID No. 40, type 2 collagen (Col2A1) promoter of sequence SEQ ID No. 41, and Prrx 1 promoter of sequence SEQ ID No.42.
According to a preferred embodiment of the invention, said vector comprises an expression cassette of sequence SEQ ID NO: 3.
According to a preferred embodiment said vector comprises a polynucleotide of sequence comprising SEQ ID NO:7.
Preferably, the vector of the invention is a viral vector, more preferably a viral vector suitable for gene therapy.
Suitable viruses for expression vectors delivery include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, baculoviruses, picornaviruses, and alphaviruses.
According to a preferred embodiment, the molecule of the invention is a viral vector for delivery of an expression vector, said expression vector comprising a polynucleotide coding for an activator of Beclin 1/Vps34 complex; said viral vector is preferably selected from the group of: adenoviral vectors, adeno-associated viral (AAV) vectors, pseudotyped AAV vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors. Pseudotyped AAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example an AAV2/8 vector contains the AAV8 capsid and the AAV 2 genome. Such vectors are also known as chimeric vectors. The present invention preferably employs adeno-associated viruses (AAV).
Exemplary AAV vectors, for use in embodiments of the present invention, include AAV types 2, 8, 9, 2/1, 2/2, 2/5, 2/7, 2/8, 2/9, rh10, rh39, rh43.
According to a preferred embodiment, a vector according to the invention may be administered to a subject in need thereof at a dose range between 1×109 viral particles (vp)/kg and 1×1014 vp/kg, a dose range between 1×1010 vp/kg and 1×1013 vp/kg, a dose range between 1×1011 vp/kg and 1×1012 vp/kg.
Naked plasmid DNA vectors and other vectors known in the art may also be used according to the present invention. Other examples of delivery systems include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection.
In the present invention polynucleotides or peptides may be isolated. A peptide according to the invention may be a recombinant peptide, obtained by any know methods in the art.
A peptide or a fragment thereof according to the invention may be synthesized via standard methods of synthetic chemistry, i.e. homogeneous chemical syntheses in solution or in solid phase. By way of illustration, those skilled in the art may use the polypeptide solution-synthesis techniques described by Houben Weil (1974, in Methode der Organischen Chemie, E. Wunsh ed., volume 15-1 and 15-11, Thieme, Stuttgart.). A peptide or a fragment thereof according to the invention may also be synthesized chemically in liquid or solid phase by successive coupling of the various amino acid residues (from the N-terminal end to the C-terminal end in the liquid phase, or from the C-terminal end to the N-terminal end in the solid phase). Those skilled in the art may especially use the solid-phase peptide synthesis technique described by Merrifield (Merrifield R. B., (1965a), Nature, vol. 207 (996): 522-523; Merrifield R. B., (1965b), Science, vol. 150 (693): 178-185).
According to another aspect, a peptide, a derivative or a fragment thereof according to the invention may be synthesized by genetic recombination in a host cell and purified, as an example, by the purification techniques described by Molinier-Frenkel (2002, J. Viral. 76, 127-135), by Karayan et al. (1994, Virology 782-795) or by Novelli et al. (1991, Virology 185, 365-376).
During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells. In the present invention the delivery vehicles may be administered to a patient. A skilled worker would be able to determine appropriate dosage range. The term “administered” includes delivery by viral or non-viral techniques. Non-viral delivery mechanisms include but are not limited to lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
The present invention also concerns pharmaceutical compositions comprising the molecule of the invention, optionally in combination with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).
Pharmaceutical compositions adapted for topical or parenteral administration, comprising an amount of a compound, constitute a preferred embodiment of the invention. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
The dose administered to a patient, particularly a human, in the context of the present invention should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.
In particular, Beclin 1 peptide or a fragment or a derivative thereof may be administered at a dose from 0.001 to 100 mg/kg of body weight, preferably from 0.01 to 50 mg/kg, still preferably from 0.1 to 10 mg/kg, yet preferably from 0.5 to 5 mg/kg, more preferably from 1 to 3 mg/kg.
mTORC inhibitors are administered at a dose from 0.001 to 100 mg/day, preferably from 0.01 to 50 mg/day, still preferably from 0.1 to 10 mg/day, yet preferably from 0.5 a 5 mg/day, more preferably from 1 a 3 mg/day.
The methods of the present invention can be used with humans and other animals. As used herein, the terms “patient” and “subject” are used interchangeably and are intended to include such human and non-human species. Likewise, in vitro methods of the present invention can be earned out on cells of such human and non-human species.
The subject invention also concerns kits comprising the molecule or vector or the host cells of the invention in one or more containers. Kits of the invention can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit of the invention includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit of the invention includes instructions or packaging materials that describe how to administer a vector system of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the molecule or vector or the host cells of the invention is provided in the kit as a solid. In another embodiment, the molecule or vector or the host cells of the invention is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing the molecule or vector or the host cells of the invention in liquid or solution form.
The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the molecule of the present invention. Preferably, the gene therapy may be achieved by the administration of a single vector comprising:
i) a polynucleotide coding for any of the molecules of the invention described herein; more preferably a polynucleotide coding for a beclin 1 derivative, more preferably a polynucleotide coding for a Tat-Beclin 1 peptide, or a retro-inverso Tat-Beclin 1 peptide, or derivatives thereof, as herein described; and
ii) a polynucleotide coding for the wild-type form of the protein whose mutated form is responsible for the bone growth disorder.
Alternatively, two vectors may be used, each comprising i) or ii), respectively.
Exemplary protein whose mutated form is responsible for a bone growth disorder include: FGFR3, FGFR1, FGFR2, β-glucocerebrosidase, α-mannosidase, α-fucosidase, α-neuraminidase, Cathepsin-A, UDP-N-acetylglucosamine, N-acetylglucosamine-1-phosphotransferase, Sulfatase modifying factor 1, Cathepsin K, α-L-iduronidase, Iduronate-2-sulfatase, Heparan N-sulfatase, α-N-acetyl glucosaminidase, Acetyl-CoA: α-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-6-sulfatase, 11-D-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, Hyaluronidase.
The pharmaceutical composition may be for human or animal usage. The vector can be administered in vivo or ex vivo.
Typically, an ordinary skilled clinician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular individual and administration route. A dose range between 1×109 and 1×1015 genome copies of each vector/kg, preferentially between 1×1010 and 1×1014 genome copies of each vector/kg, more preferentially from 1×1011 and 1×1013 are expected to be effective in humans. A preferred dose is 4,5×1012 genome copies of each vector/kg.
Dosage regimes and effective amounts to be administered can be determined by ordinarily skilled clinicians. Administration may be in the form of a single dose or multiple doses. General methods for performing gene therapy using polynucleotides, expression constructs, and vectors are known in the art (see, for example, Gene Therapy: Principles and Applications, Springer Verlag 1999; and U.S. Pat. Nos. 6,461,606; 6,204,251 and 6,106,826).
The molecules according to the invention can activate the Beclin 1/Vps34 complex either directly, e.g. by interacting with the complex, or indirectly, e.g. by interacting with molecules regulating the complex.
In a further aspect, the invention provides a composition comprising the molecule according to any one of previous claims and pharmaceutically acceptable excipients for use in the treatment of a bone growth disorder.
Preferably the composition further comprises a wild-type form of a protein, whose mutated form is responsible for a lysosomal storage disorder with skeleton involvement; preferably said protein is selected from the group consisting of FGFR3, FGFR1, FGFR2, FGFR4, β-glucocerebrosidase, α-mannosidase, α-fucosidase, α-neuraminidase, Cathepsin-A, UDP-N-acetylglucosamine, N-acetylglucosamine-1-phosphotransferase, Sulfatase modifying factor 1, Cathepsin K, α-L-iduronidase, Iduronate-2-sulfatase, Heparan N-sulfatase, α-N-acetyl glucosaminidase, Acetyl-CoA: α-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-6-sulfatase, β-D-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, Hyaluronidase. Still preferably the composition further comprises a polynucleotide comprising a nucleotide sequence encoding for a wild-type form of said protein whose mutated form is responsible for said lysosomal storage disorder with skeleton involvement.
In a further aspect the invention provides a method of treatment of bone growth disorder comprising administering to a subject in need thereof a molecule as defined above or a composition as defined above or a vector as defined above.
According to preferred embodiments of the invention the bone growth disorder is selected from the group consisting of: achondroplasia, hypochondroplasia, MPS I, MPS II, MPS IV, MPS VI, MPS VII, MPS IX, Gaucher disease type 3, Gaucher disease type 1, a glycoproteinoses, multiple sulfatase deficiency, a pycnodysostosis and a spondyloepiphyseal dysplasia; more preferably, the bone growth disorder is selected from the group consisting of achondroplasia, MPS VI, MPS VII.
Tat-Beclin 1 peptide is capable of inducing autophagy in a cell by activating Beclin 1-Vps34 complex (see FIG. 4A1).
Daily injection of Tat-Beclin1 peptide promoted Av-Lys fusion and p62/SQSTM1 degradation in the growth plate of MPS VII (Gusb−/−) mice expressing the fluorescent autophagy reporter GFP-LC364 (Gush−/−; GFP-LC3tg/+ mice) (
Newborn MPS VII and MPS VI mice were intraperitoneally injected daily with retro-inverso Tat-Beclin 1 peptide (Beclin 1Activator II, retro-inverso Tat-Beclin 1, Millipore) at 2 mg/kg resuspended in PBS, according to a preferred embodiment of the invention. Control mice were injected with vehicle only. Mice were sacrificed after 15 (P15) and 30 (P30) days.
Starting at postnatal day 15 (P15) MPSVII mice show significant reduced femur and tibia lengths compared to wild type mice (
RCS cells stably expressing FGFR3 wild-type (wt), R248C (FGFR3TD) and G380R (FGFR3ach) mutations, associated to achondroplasia in humans, were prepared by retroviral transduction. FGR3ach and FGFRTD chondrocytes were treated with lysosomal inhibitors leupeptin and bafilomycin to clamp autophagosomes (AVs) degradation. Leupeptin and bafilomycin treatments did not increase the level of LC3II protein in FGR3ach and FGFRTD chondrocytes, compared to FGFR3 wild type stable cells (
Animals: MPSVI (Arsb−/−) mice59,50 obtained from A. Auricchio (Telethon Institute of Genetics and Medicine, TIGEM, Naples). MPSVII mice (Gusb−/−)58 were obtained from Jackson Laboratories. All mice used were maintained in a C57BL/6 strain background. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health. Tissues and histology: Histology was performed according to standardized procedures (http://empress.har.mrc.ac.uk/browser/). Briefly, femurs were fixed ON in 4% (wt/vol) paraformaldehyde (PFA) and then demineralized in 10% EDTA (pH 7.4) for 48h. Specimens were then dehydrated, embedded in paraffin and sectioned at 7 μm, and stained with hematoxylin and eosin. For BrDU staining mice were injected with 200 μl of 10 mM BrDU (Sigma) 4h before sacrifice. BrDU incorporation was detected using a Zymed BrDU staining kit (Invitrogen). Counterstaining was performed using hematoxylin Immunohistochemistry were performed according to standardized protocols. Briefly, type X collagen (Hybridoma bank) staining were performed pretreating paraffin-embedded sections with 1 mg/ml pepsin in 0.1 M Acetic Acid, 0.5 M NaCl for 2 h at 37° C., and then treated with 2 mg/ml hyaluronidase in 0.1 M TBS for 1 h at 37° C., prior to the blocking step. Endogenous peroxidases were quenched with 3% hydrogen peroxide, sections were then incubated with blocking serum and primary antibody over night at 4° C. Signals were developed using Vectastain Elite ABC kit (Vector Laboratories) and NovaRED Peroxidase Substrate kit (Vector Laboratories).
Western blotting: Cells were washed twice with PBS and then scraped in lysis buffer (RIPA lysis buffer in the presence of PhosSTOP and EDTA-free protease inhibitor tablets—Roche, Indianapolis, Ind., USA). Cell lysates were incubated on ice for 20′, then the soluble fraction was isolated by centrifugation at 14,000 rpm for 10 mM at 4° C. Total protein concentration in cellular extracts was measured using the colorimetric BCA protein assay kit (Pierce Chemical Co, Boston, Mass., USA). Protein extracts, separated by SDS-PAGE and transferred onto PVDF membrane, were probed with antibodies against LC3, β-actin (Novus Biologicals), P62 (Abnova) and FGFR3 (Cell Signaling). Proteins of interest were detected with HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:2000, Vector Laboratories) and visualized with the Super Signal West Dura substrate (Thermo Scientific, Rockford, Ill.), according to the manufacturer's protocol. The Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using imageJ software using “Gels and Plot lanes” plug-in.
Retrovirus preparation: Retroviral particles were produced using packaging plasmids (VSV-G and gag/pol) (Addgene) in 293T cells (ATCC, Manassas, Va.). 293T were cultured in DMEM containing 10% FBS and were transfected using Lipofectamine LTX and Plus reagent (Invitrogen). The supernatant containing retroviral particles was collected after 48-72 hours for RCS transduction and filtered through 0.45 mm filter (Corning). Infected RCS cells were selected with puromycin (2.5 μg/mL). Plasmids: pBp-FGFR3c-wt and pBp-FGFR3c-R248C were purchased from Addgene; pBp-FGFR3c-G380R was generated using QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies).
Leupeptin and bafilomycin treatments: Leupeptin (Sigma) was resuspended in water at 10 mM. FGFR3 wild type, FGFR3ach and FGFR3TD stable chondrocytes cell line were treated with 50 μM leupeptin for 2 h at 37° C. Bafilomycin (Millipore) was resuspended in DMSO at 200 μM. FGFR3 wild type, FGFR3 ach and FGFR3TD stable chondrocytes cell line were treated with 200 nM bafilomycin for 4 h at 37° C.
FACS: RCS cells stably expressing FGFR3 WT, R248C and G380R were harvested in trypsin, washed with PBS, fixed for 10 mM in ice-cold methanol and permeabilized for 15 min with 100 μg/mL digitonin in PBS. Cells were then incubated with mouse anti-LC3 primary antibody (Nanotools) for 30 min, washed three times in PBS, and incubated for 30 mM with goat anti-mouse secondary antibody (Alexa labelled). FACS data were collected using BD Accuri C6 Cytometer (BD Biosciences) and data analysis was carried out with BD Accuri C6 Software.
The femoral growth plates of mice that ubiquitously express the autophagosome marker MAP1LC3 tagged with green fluorescent protein (GFP) (GFP-LC3tg/+) (Mizushima N et al, Mol Biol Cell 2004) were analyzed. Very few autophagic vesicles (AVs) were detected in the growth plates of newborn mice (P0) (
The essential autophagy gene 7 (Atg7) was deleted in chondrocytes by crossing a mouse line carrying the Atg7 floxed allele (Atg7f/f) (Komatsu, M. et al., J. Cell Biol. 2005) with two different Cre mouse lines: 1) the Prx1-Cre line, in which the Cre protein is expressed in the mesenchymal cells of the limbs during embryogenesis (Logan M et al., Genes 2002) and 2) the Col2a1-Cre line, in which the expression of the Cre protein is mainly restricted to mature chondrocytes before and after birth (Ovchinnikov DA, Genes 2002).
The selective lack of Atg7 protein and the inhibition of functional autophagy in the femoral growth plates of Atg7f/f; Prx1-Cre and Atg7f/f; Col2a1-Cre mice was verified (
Atg7f/f; Prx1-Cre and Atg7f/f; Col2a1-Cre mice were born at the expected Mendelian ratio, with bones of normal shapes and sizes, suggesting that chondrocyte autophagy is dispensable during embryonic skeletal development (
Histological analyses of femoral and tibia growth plates from P6 and P9 Atg7f/f; Prx1-Cre and Atg7f/f; Col2a1-Cre mice showed preserved architecture and normal rates of chondrocyte differentiation, proliferation and terminal apoptosis, suggesting that these processes occur independently of autophagy in chondrocytes (
The levels of glycosaminoglycans, were only slightly reduced in the growth plate of Atg7f/f; Prx1-Cre and Atg7f/f; Col2a1-Cre mice compared to controls (
By using an antibody that recognizes the pro-alpha1(II) chain of Col2/PC2 proteins (Col2a1), accumulation of Col2a1 molecules in the ER of chondrocytes lacking autophagy (
The inhibition of autophagy with Spautin-1 (Liu, J. et al., Cell 2011) or with RNA interference targeting Atg7(Atg7Kd) in cultured Rx chondrocytes in which PC2 secretion was synchronized (Venditti R et al., Science 2012) led to defective PC2 secretion and to the retention of PC2 in the ER (
The presence of PC2 in at least 15% of the AVs analyzed (
Dual-color (mCherry-PC2 and GFP-LC3) live cell imaging experiments using Rx chondrocytes in which PC2 secretion was synchronized showed the selective sequestration of PC2 aggregates by GFP-LC3 positive vesicles (
Furthermore, the 47 kDa collagen-specific chaperone HSP47, which associates to native PC2 triple helices in the ER and mediates their ER to cis-Golgi trafficking15, was excluded from the AVs containing PC2, suggesting that autophagy selectively recognizes non-native PC2 molecules in the ER (
In addition, Spautin-1 treatment inhibited the ER to cis-Golgi trafficking of HSP47 in cultured chondrocytes (
During autophagy, AVs target their cargo to lysosomes. Consistently dual-color (mCherry-PC2 and GFP-LAMP1) live cell imaging experiments showed progressive and autophagy-dependent accumulation of PC2 in GFP-LAMP1 vesicles (
These data show that autophagy is required for PC2 homeostasis and secretion, rather than directly mediating PC2 exocytosis (
Primary chondrocytes isolated from GFP-LC3 mice were stimulated with FGF18 and other chondrogenic factors (Karsenty, G et al., Annu. Rev. Cell Dev. Biol. 2009) and autophagosome biogenesis was assessed in the presence of BafA1 (
Most importantly, in vivo studies revealed that autophagy is completely inhibited in the growth plates of Fgfl 8−/− E18.5 embryos, as demonstrated by undetectable levels of LC3II and accumulation of the autophagy receptor P62/SQSTM1 compared to control mice (
Fgfl 8−/− mice exhibit neonatal lethality (Liu Z et al. Genes Dev 2002), therefore the growth plates of Fgfl 8+/− mice, during early post-natal development, were analyzed: the levels of autophagy were similar in newborn Fgf18+/− and control mice, but the subsequent post-natal induction of autophagy was abrogated in Fgfl 8+/− mice (
Sections of growth plates isolated from P6 Fgfl 8+/−; GFP-LC3tg/+ mice had significantly fewer GFP-labeled AVs compared to sections isolated from control Fgfl 8+/+; GFP-LC3tg/+ mice (
Leupeptin treatment significantly increased LC3II levels in the growth plate of P6 control but not in Fgfl 8+/− mice suggesting reduced AV biogenesis in Fgfl 8+/− chondrocytes (
RNA interference of either Fgfr3 or Fgfr4, but not Fgfr1 and Fgfr2, inhibits FGF18-induced autophagy in Rx chondrocytes (
Canonical FGF signaling activates the mitogen-activated protein kinase (MAPK) pathway. The growth plates of Fgfl 8+/− mice show lower levels of JNK1/2 kinase activation than control mice (
Active JNK1 phosphorylates Bcl2 and disrupts the Bcl2-Beclin 1 complex (Wei Y et al., Mol Cell 2008), leading to the activation of the Class III PI 3-kinase Vps34/Beclin 1 complex, which produces the phosphatidylinositol 3-phosphate (PI3P) required for AV biogenesis (Liang X H et al., Nature 1999).
FGF18 increases the phosphorylation of Bcl-2 in a JNK-dependent manner (
Enhancing Beclin 1 activity by intraperitoneal (IP) injection of a synthetic Tat-Beclin 1 peptide, as defined herein, normalizes autophagy levels in the growth plates of Fgf18+/−; GFP-LC3tg/+ mice (
Rx chondrocytes stimulated with FGF18 exhibited higher efficiency of PC2 secretion compared to non-stimulated cells, but addition of the autophagy inhibitor Spautin-1 hampered this increase (
Therefore, the growth plate phenotype of Fgf18+/− mice mimics the one observed in mice lacking autophagy in chondrocytes. Notably, the few GFP-labeled AVs detectable in the growth plates of Fgfl 8+/−; GFP-LC3tg/+ mice contained PC2, further demonstrating that PC2 is an autophagy substrate in vivo (
Strikingly, Tat-Beclin 1 treatment restored Colt levels in the growth plates of Fgfl 8+/− mice (
In addition, Tat-Beclin 1 treatment restored Colt levels and rescued femoral growth retardation in P9 Fgfr4−/− mice (
A vector for the expression of a Beclin 1 derivative peptide, according to preferred embodiments of the invention, was prepared by conventional means. The vector comprises a cassette having sequence SEQ ID NO:3, (
Animals: The Atg7 Fe and the GFP-LC36 mouse lines were obtained from N. Mizushima (Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Japan). The Prx-1 Cre line9 was purchased from Jackson Laboratories (strain n. 005584). Col2a1-Cre line was obtained from B. Lee (Baylor College of Medicine, Houston,). The fgfl 822 and fgfr322 KO line was a generous gift from D. Ornitz (Washington University, St. Louis). The fgfr4 was obtained from Dr. Seavitt (Baylor College of Medicine, Houston, Tex.). All mice used were maintained in a C57BL/6 strain background. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health.
The vector plasmid for Beclin 1 derivative peptide expression used in the examples was generated as follows: sFlt1-Tat-beclin1 sequence (sFlt1 is SEQ NO.6, Tat-Beclin 1 is SEQ No.7) was synthesized de novo and was cloned into a plasmid backbone which derived from the pAAV2.1 plasmid [Auricchio A, Hildinger M, O'Connor E, Gao G P, Wilson J M (2001)] Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Hum Gene Ther 12: 71-76] and contained: the inverted terminal repeats (ITRs) of AAV serotype 2, the CMV promoter, the 3×flag tag, the WPRE and the BGH polyA. The vector plasmid was transfected into HEK293 cells using the calcium phosphate method. 24 hours later, Tat-Beclin 1 conditioned medium from transfected cells was harvested and added to a new plate of HEK293 cells. HEK293 cells were then incubated with conditioned media for 24 hours and finally harvested for Western Blot analysis.
Skeletal staining: Skeletons were fixed in 95% ethanol overnight (ON) and stained with alcian blue and alizarin red according to standardized protocols (http://empress.har.mrc.ac.uk/browser/). Three to five mice of each genotype were analyzed per stage. Measurement of bone length was performed using ImageJ software.
Tissue histology, immunohistochemistry and immunofluorescence: Histology was performed according to standardized procedures (http://empress.har.mrc.ac.uk/browser/). Briefly, femurs were fixed ON in 4% (wt/vol) paraformaldehyde (PFA) and then demineralized in 10% EDTA (pH 7.4) for 48h (demineralization was performed only if specimens were isolated from mice older than P5). Specimens were then dehydrated, embedded in paraffin and sectioned at 7 μm, and stained with hematoxylin and eosin. For BrdU staining mice were injected with 100 μL of 10 mM BrdU (Sigma) 4h before sacrifice. BrdU incorporation was detected using a Zymed BrdU staining kit (Invitrogen). The TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed using the In situ Cell Death Detection kit (Roche). Counterstaining was performed using hematoxylin. For immunofluorescence, femurs were dissected from euthanized mice and fixed with buffered 4% PFA ON at 4° C., then washed with PBS and cryoprotected in successive sucrose solutions diluted with PBS (10% for 2 hours, 20% for several hours and 30% ON at 4° C.; all wt/vol), and finally embedded in OCT (Sakura). Cryostat sections were cut at 10 μm. Sections were blocked and permeabilized in 3% (wt/vol) BSA, 5% fetal bovine serum in PBS+0.3% Triton X-100 for 3 h and then incubated with the primary antibody ON. Sections were washed three times with 3% BSA in PBS+0.3% Triton X-100 and then incubated for 3 h with secondary antibodies conjugated with Alexa Fluor 488, or Alexa Fluor 568. The extracellular Col2a1 staining was performed by pretreating sections with chondroitinase ABC (Sigma) at 0.2 U/ml for 1 h at 37° C. prior to the blocking step. Intracellular Col2a1 staining was performed without chondroitinase ABC pretreatment to stain only the Col2a1 molecules that were not masked by proteoglycans. Primary antibodies used were: GFP, Lamp1 and HSP47 (Abeam), Col2a1 (1:30, Hybridoma Bank, II6B3), VapA, Sec31, Giantin, GM130, P115, Calreticulin were previously described 13. Nuclei were stained with DAPI and sections were mounted with vectashield (Vector laboratories). Images were captured using a Zeiss LSM700 confocal microscope. Colocalization analysis was performed calculating Mander's coefficient using ImageJ (colocalization analysis plug in).
Collagen Quantification and Analysis: Colorimetric assay was performed using the Sircol soluble collagen assay (Biocolor, UK) following the manufacturer's protocol. Briefly, femural and tibial cartilages were microdissected and collagen was acid pepsin extracted and complexed with Sircol dye. Absorbance was measured at 555 nm and concentration was calculated using a standard curve. Values were normalized to DNA levels calculated measuring the absorbance at 260 nm.
Electrophoretic analysis: Three femural cartilages were isolated from mice with the same genotype, pooled and homogenized in 0.5 ml of 1 mg/ml cold (4° C.) pepsin in 0.2 M NaCl, 0.5 M acetic acid to pH 2.1 with HCl and then digested at 4° C. for 24 hours, twice. The pellet was discarded and an equal volume (1 ml) of 4 M NaCl in 1 M acetic acid was added to precipitate collagen. The pellet was then resuspended in 0.8 ml of 0.2 M NaCl in 0.5 M acetic acid and was precipitated again three times. After the last precipitation the pellet was washed twice with 70% Et-OH in order to remove residual NaCl. The pellet was then dissolved in 0.8 ml 0.5 M acetic acid, and lyophilized. Subsequently it was resuspended in Laemmli buffer without Et-SH at a concentration of 2 mg/ml, denatured at 80° C. for 5 min and loaded on 6% SDS-PAGE. Gels were then stained with Coomassie Brilliant Blue R-250.
GAG quantification: GAG quantification was performed using the Blyscan sulfated glycosaminoglycan assay (Biocolor, UK) following the manufacturer's protocol. Briefly, femural and tibial cartilages were microdissected and GAGs were papain extracted at 65° C. ON and complexed with Blyscan dye. Absorbance was measured at 656 nm and concentration was calculated using standard curve. Values were normalized to DNA levels calculated measuring the absorbance at 260 nm.
Transmission electron microscopy: For EM analysis growth plates were fixed in 1% glutaraldehyde in 0.2M HEPES buffer. Small blocks of growth plates were then post-fixed in uranyl acetate and in OsO4. After dehydration through a graded series of ethanol, tissue samples were cleared in propylene oxide, embedded in Epoxy resin (Epon 812) and polymerized at 60° C. for 72h. From each sample, thin sections were cut with a Leica EM UC6 ultramicrotome and images were acquired using a FEI Tecnai-12 (FEI, Einhoven, The Netherlands) electron microscope equipped with Veletta CCD camera for digital image acquisition.
Tat-Beclin 1 peptide and Leupeptin treatment: Newborn mice were intraperitoneally injected daily with Tat-Beclin 1 peptide (Beclin 1 Activator II, retro-inverso Tat-Beclin 1, Millipore) at 20 mg/kg resuspended in PBS25. Control mice were injected with vehicle only. Mice were sacrificed after 6 days (Col2a1 IF experiments) or 9 days (total collagen quantification). Leupeptin (Sigma Cat. L2884) was resuspended in water at 10 mM. Mice were give intraperitoneal injection at 40 mg/kg. Six hours after injection tissues were harvested and processed.
Tissue protein extracts for Western blotting: Femural and tibia cartilages were microdissected and lysed using a tissuelyser (Qiagen) in RIPA lysis buffer supplemented with 0.5% SDS, PhosSTOP and EDTA-free protease inhibitor tablets (Roche, Indianapolis, Ind., USA). Samples were incubated for 30 min on ice, briefly sonicated on ice and the soluble fraction was isolated by centrifugation at 14,000 rpm for 10 min at 4° C.
Chemicals: FGF18 (50 ng/ml), PTHrP (10 μg/ml), BMP2 (500 ng/ml) were from Peprotech, rhSHH (10 μg/ml) from R&D Systems. c-Jun N-Terminal kinase (INK) inhibitor (SP600125, Sigma-Aldrich, Milan, Italy) (50 μM) was used for the indicated time. Tannic Acid (Fluka chemika) was used at 0.5% final concentration in the medium for 1 h at 37° C. Bafilomycin A1 (Sigma) was used at 200 nM.
Cell Culture, transfections, SiRNA and Plasmids: Primary cultured chondrocytes were prepared from rib cartilage of P5 mice. Rib cages were first incubated in DMEM using 0.2% collagenase D (Roche) and after adherent connective tissue had been removed (1.5 h) the specimens were washed and incubated in fresh collagenase D solution for a further 4.5 h. Isolated chondrocytes were maintained in DMEM (Gibco) supplemented with 10% FCS and ascorbic acid (50 mg ml−1). Since an incomplete deletion of the Atg7 gene in Atg7f/f; Col2a1-Cre growth plates was observed (
Live cell imaging: Rx chondrocytes were reverse transfected and plated in Mattek glass bottomed dishes. Collagen transport assays were performed by incubating cells at 40° C. on the heated stage for 2.5 h. Collagen release was initiated by lowering the temperature of the stage to 32° C. and medium being supplemented with 50 μg/ml ascorbate.
TIRF: Rx chondrocytes were reverse transfected and plated in Mattek glass bottomed dishes. Rx cells were synchronized on the heated stage for 2.5 h at 40° C. and released at 32° C., in medium supplemented with 50 μg/ml ascorbate in a humidified atmosphere with 5% CO2. The critical angle used was 65 degrees giving an evanescent field of 137 nm. Appropriate filter sets were used for GFP and mCherry detection. Frames were acquired on loop with no time delay (one frame roughly every 3s), for 15 min. All live cell imaging experiments was performed with a 60× Plan Apo oil immersion lens using a Nikon Eclipse Ti Spinning Disk microscope, and images and movies were annotated using the NIS Elements 4.20 software. Western blotting: Cells were washed twice with PBS and then scraped in lysis buffer (RIPA lysis buffer in the presence of PhosSTOP and EDTA-free protease inhibitor tablets—Roche, Indianapolis, Ind., USA). Cell lysates were incubated on ice for 20′, then the soluble fraction was isolated by centrifugation at 14,000 rpm for 10 min at 4° C. Total protein concentration in cellular extracts was measured using the colorimetric BCA protein assay kit (Pierce Chemical Co, Boston, Mass., USA). Protein extracts, separated by SDS-PAGE and transferred onto PVDF or nitrocellulose (for collagen) membranes, were probed with antibodies against P-JNK, JNK, P-Bcl-2, P-c-JUN (Cell Signaling Technology), HA, H3 Histone (Sigma-Aldrich, Milan, Italy) and LC3 (Novus Biologicals), p62 (BD Transduction Laboratories and Abnova), PDI (Cell Signaling), GOLPH3 (Abeam), p-ERK, ERK1/2 (Cell Signaling), p-P38, P38 (Cell Signaling), Beclin 1 (Cell Signaling), VPS34 (Sigma-Aldrich, Milan, Italy), b-actin (Novus Biologicals), GAPDH (Santa Cruz Biotecnology), Atg7 (Cell Signaling), p-mTORC1, mTORC1 (Cell Signaling), p-P70S6K, P70S6K (Cell Signaling), p-4EBP1, 4EBP1 (Cell Signaling), p-AKT, AKT (Cell Signaling), p-AMPKa, AMPKa (Santa Cruz Biotecnology), type II collagen (CIIC1b,Hybridoma Bank). Proteins of interest were detected with HRP-conjugated goat antimouse or anti-rabbit IgG antibody (1:2000, Vector Laboratories) and visualized with the Super Signal West Dura substrate (Thermo Scientific, Rockford, Ill.), according to the manufacturer's protocol. The Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using imageJ software using “Gels and Plot lanes” plug-in.
High content screening analysis in GFP-LC3 primary chondrocytes: Primary chondrocytes were plated in CellCarrier-96 Black plates (6005558, Perkin Elmer). After identifying the nuclei with Hoechst 33342 (405 nm) staining, a cytoplasmic mask was drawn using Col2 staining (568 nm). To carry out the analysis the number of cytoplasmic GFP-LC3 spots in the cytoplasm of Col2 positive cells were counted, and expressed per cell. Levels of colocalization between GFP-LC3 and Col2a1 were assessed and expressed as %, using the parameters: area of colocalization of red spots with area of green spots normalized to total area of green spots. Image acquisition was performed using Opera High Content Screening System (PerkinElmer); image analysis was performed using Acapella High Content Imaging and Analysis Software (PerkinElmer). For GFP-LC3 puncta count, at least 1000 cells were analyzed for each treatment from 3 independent chondrocyte preparations. Repeated measures ANOVA was performed with TUKEYs post-hoc test. For GFP-LC3/col2a1 colocalization, at least 700 cells were analyzed per field from 2 different chondrocyte preparations.
Co-immunoprecipitation: Rx chondrocytes (100-mm dish) were grown in DMEM medium (Celbio, Milan, Italy) with 10% fetal bovine serum (FBS—Invitrogen corporation, Carlsbad, Calif., USA) and antibiotics. For FGF18 treatment, 70 to 80% confluent cells were cultured ON in DMEM with 10% adult bovine serum (Sigma-Aldrich, Milan, Italy) and then treated with FGF18 (50 ng/ml, 2 h) (Peprotech, Ottawa, Ontario) or DMSO vehicle. Rx chondrocytes were rinsed off the plate with ice-cold PBS, washed, and then scraped in IP lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40, with one PhosSTOP and one EDTA-free protease inhibitor tablet per 10 ml—Roche, Indianapolis, Ind., USA). Cell lysates were rotated at 4° C. for at least 30 min, and then the soluble fraction was isolated by centrifugation at 14,000 rpm for 10 mM at 4° C. A fraction of the clarified lysate was used for Western blot analysis. Primary Beclin 1 (H-300) rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, Calif.) antibody or rabbit pre-immune IgG were added to the lysates and rotated over night at 4° C., and then 25 μl of Protein A Sepharose beads (Sigma-Aldrich, Milan, Italy) were added and rotated for 2 h at 4° C. Immunoprecipitates were washed 3 times with cold lysis buffer. Whole cell lysates and immunoprecipitated proteins were boiled in 30 μl sample buffer, separated by SDS-PAGE on precast 4-15% gels (BioRad), transferred on PVDF membranes and probed with antibodies against Beclin 1 (Santa Cruz Biotechnology, Santa Cruz, Calif.), VPS34 (Sigma-Aldrich, Milan, Italy) and Bcl-2 (Cell Signaling Technology).
PI3K assay: PI3K activity in the Beclin 1 immunoprecipitates was determined using the PI3K ELISA kit (Echelon Biosciences, Inc., Salt Lake City, Utah) according to the manufacturer's instructions Immunocomplexes were incubated with a reaction mixture containing PtdIns(4,5)P2 substrate and ATP for 3 hours, and the amount of PtdIns(3,4,5)P3 generated from phosphatidylinositol 4,5-bisphosphate by PI3K was quantified using a competitive ELISA. Equal amounts of Beclin 1 immunoprecipitate were evaluated by Western blotting using Beclin 1 antibody.
Cell immunofluorescence: Chondrocytes were fixed for 10 min in 4% PFA in PBS and permeabilized for 30 min in 0.05% (w/v) saponin, 0.5% (w/v) BSA, 50 mM NH4Cl and 0.02% NaN3 in PBS (blocking buffer). The cells were incubated for 1 h with the primary antibodies, washed three times in PBS, incubated for 1 h with the secondary (Alexa fluor-labeled) antibody, washed three times in PBS, incubated for 20 min with 1 μg/ml Hoechst 33342 and finally mounted in Mowiol. All confocal experiments showing colocalization were acquired using slice thickness of 0.5 mm using the LSM 710 confocal microscope equipped with a 63×1.4 numerical aperture oil objective.
Procollagen secretion assay: To follow PC2 secretion in Rx chondrocytes, cells were pretreated ON with ascorbate (100 μg/ml) in DMEM without FCS. Cells were then labeled with 37.5 μCi/mL 2,3 3H-Proline (Perkin Elmer) for 4 h at 40° C. in the same medium then shifted to 32° C. in DMEM without FCS containing cold proline (10 mM), 20 mM HEPES pH 7.2 and ascorbate (100 μg/ml). After 0, 30 and 60 minutes the medium and cells were collected, lysed and proteins precipitated in saturated ammonium sulfate ON and resuspended in Laemmli buffer. Samples were run on 4-15% precast gels (Biorad), transferred onto nitrocellulose membrane (Whatman, Perkin Elmer) and developed by autoradiography using the BetaIMAGER-D system and analyzed using M3 Vision software (Biospace Lab).
Primary chondrocytes were isolated from the rib cage of post-natal day 5 mice (wild-type and MPS VII) and were plated at the density of 105 cells/cm2. After 3 days in culture cells were splitted in 12 wells chamber for biochemical analysis (
Primary chondrocytes isolated from chondrocostal cartilage of newborn MPS VII mice show prominent lysosomal storage phenotype characterized by cytoplasm filled with giant lysosomes, which were undetectable in chondrocytes isolated from control littermates (
The mTORC1 kinase promotes anabolic processes, such as protein and lipid synthesis, in response to nutrients and growth factors stimulation55. In addition, mTORC1 regulates lysosome/autophagy and proteasome functions through both transcriptional and post-translational mechanisms53,56. Thus, mTORC1 controls the cellular balance between catabolic and anabolic metabolisms in response to nutrient levels.
Major regulators of mTORC1 are amino acids that can be either supplied with the diet or de-novo synthesized starting from metabolic intermediates57. In addition the amino acid pool produced by lysosome and proteasome-mediated protein catabolism can also influence mTORC1 signaling54. However, the physiological relevance of this source of amino acids as regulator of mTORC1 activity is still largely unknown.
Primary chondrocytes were isolated from the rib cage of P5 mice (wild-type and MPS VII) and were plated at the density of 105 cells/cm2. After 3 days in culture cells were splitted in 12 wells chamber for biochemical analysis (
The activity of mTORC1 was analyzed in mouse primary chondrocytes and in a RCS chondrocytes isolated from mouse models of MPSVII (Gusb−/−)58 and MPSVI (Arsb−/−)59, mesenchymal-derived chondrocytes60 isolated from three MPSI61 human patients and RCS model of MPSVII (GusbKO) generated by Crisp/Cas9 technology, all showed enhanced mTORC1 signaling (n enhanced phosphorylation of p70 S6 Kinase and of ULK1) in response to amino acid stimulation compared to their correspondent controls (
The response of MPS VII chondrocytes to growth factor (FBS 10%) stimulation was similar to that observed in control cells (
MPS chondrocytes showed a severe lysosome phenotype as demonstrated by enlarged Lys filled with undigested substrates and accumulation of the lysosomal marker LAMP1. In addition, the inventors also observed a significant accumulation of AVs, as demonstrated by increase number of LC3 positive vesicles and accumulation of the autophagosome-associated form of MAPLC3B protein (LC3II) (
Without being bound to theory, enhanced activity of mTORC1 could be a consequence of increased association with lysosomes in MPS VII cells.
Primary chondrocytes were isolated from the rib cage of P5 mice (wild-type and MPS VII) and were plated at the density of 105 cells/cm2. After 3 days in culture cells were splitted in 12 wells chamber, synchronized with AA, treated with Torin1 (1 μM) for 24 hours and harvested for biochemical analysis.
Pharmacological inhibition of mTORC1 with Torin1 completely suppresses phosphorylation of mTORC1 substrates and rescues the autophagy defects in MPSVII chondrocytes, as demonstrated by normalization of LC3 II and p62 levels (
These data indicate that normalization of mTORC1 signaling is sufficient to ameliorate the cellular phenotype in MPS VII chondrocytes, indicating that mTORC1 dysfunction could account, at least in part, for the autophagy defects in MPS VII chondrocytes.
Raptor (RPT or Gusb−/−; Rpt+/−) mice are MPS VII mice (Gusb−/−) carrying only one functional copy of raptor allele (
Genetic limitation of mTORC1 rescues the altered signaling found in MPSVII chondrocytes, thus RPT cells show 20% reduction in the levels of both P-ULK1 and P-p70S6K activation (
mTORC1 can inhibit AV-Lys fusion by phosphorylation of UV radiation resistance-associated gene (UVRAG) protein, enhancing its affinity for the inhibitor partner Rubicon. Several lines of evidence suggested that this was the case in MPS chondrocytes: GusbKO cells had higher levels of UVRAG serine 497 (S497) phosphorylation compared to control cells, and this phosphorylation was blunted by the mTOR inhibitor Torin-1 (
WT, MPS VII and RPT littermates were sacrificed at post-natal day 15. Four hours before sacrifice mice were injected with BrdU at 0.1 mg/g body weight. Skeletons were prepared and stained with Alizarin Red/Alcian Blue. For analyses on sections limbs were collected, decalcified, processed and sectioned in paraffin.
Skeletal preparations showed that removing one allele of raptor rescued MPS VII mice short stature at post-natal day 15, as determined by femur and tibia length (A). Importantly this rescue is maintained up to post-natal day 30 (
Consistently histological analysis of femur and tibia sections showed that chondrocyte proliferation measured by BrdU incorporation, which was significantly reduced by 7% in the P15 MPS VII, was indistinguishable from wild-type in RPT P15 mice (
Skeletal staining: Skeletons were fixed in 95% ethanol overnight (ON) and stained with alcian blue and alizarin red according to standardized protocols (http://empress.har.mrc.ac.uk/browser/). Three to five mice of each genotype were analyzed per stage. Measurement of bone length was performed using ImageJ software.
Tissues and histology: Histology was performed according to standardized procedures (http://empress.har.mrc.ac.uk/browser/). Briefly, femurs were fixed ON in 4% (wt/vol) paraformaldehyde (PFA) and then demineralized in 10% EDTA (pH 7.4) for 48h. Specimens were then dehydrated, embedded in paraffin and sectioned at 7 μm, and stained with hematoxylin and eosin. For BrDU staining mice were injected with 200 μl of 10 mM BrDU (Sigma) 4h before sacrifice. BrDU incorporation was detected using a Zymed BrDU staining kit (Invitrogen). Counterstaining was performed using hematoxylin Immunohistochemistry were performed according to standardized protocols. Briefly, type X collagen (Hybridoma bank) staining were performed pretreating paraffin-embedded sections with 1 mg/ml pepsin in 0.1 M Acetic Acid, 0.5 M NaCl for 2 h at 37° C., and then treated with 2 mg/ml hyaluronidase in 0.1 M TBS for 1 h at 37° C., prior to the blocking step. Endogenous peroxidases were quenched with 3% hydrogen peroxide, sections were then incubated with blocking serum and primary antibody over night at 4° C. Signals were developed using Vectastain Elite ABC kit (Vector Laboratories) and NovaRED Peroxidase Substrate kit (Vector Laboratories).
Cell Culture: Primary chondrocytes were isolated from the rib cage of post-natal day 5 mice. Rib cages were first incubated in DMEM using 0.2% collagenase D (Roche) and after adherent connective tissue had been removed (1.5 h) the specimens were washed and incubated in fresh collagenase D solution for a further 4.5 h. Isolated chondrocytes were maintained in DMEM (Gibco) supplemented with 10% FCS and were plated at the density of 105 cells/cm2. After 3 days in culture cells were splitted in 12 wells chamber for biochemical analysis (Western blot) or on cover slips for immunofluorescence analysis. For amino acid stimulation cells were starved 1h in RPMI-1640 medium (USbio) without amino acids and supplemented with 10% dialyzed FBS (Invitrogen, Life Technologies) then cells were treated for the indicated time-points with a mixture of essential amino acids, non-essential amino acids and L-glutammine (Invitrogen, Life technologies) at the final concentration of 3×.
Western blotting: Cells were washed twice with PBS and then scraped in lysis buffer (RIPA lysis buffer in the presence of PhosSTOP and EDTA-free protease inhibitor tablets—Roche, Indianapolis, Ind., USA). Cell lysates were incubated on ice for 20′, then the soluble fraction was isolated by centrifugation at 14,000 rpm for 10 min at 4° C. Total protein concentration in cellular extracts was measured using the colorimetric BCA protein assay kit (Pierce Chemical Co, Boston, Mass., USA). Protein extracts, separated by SDS-PAGE and transferred onto PVDF or nitrocellulose (for collagen) membranes, were probed with antibodies against P-ULK(5757), ULK1, P-p70S6K (T389), p70S6K (Cell Signaling Technology), LC3 (Novus Biologicals), p62 (BD Transduction Laboratories and Abnova), b-actin (Novus Biologicals), LAMP1 (Abacam). Proteins of interest were detected with HRP-conjugated goat antimouse or anti-rabbit IgG antibody (1:2000, Vector Laboratories) and visualized with the Super Signal West Dura substrate (Thermo Scientific, Rockford, Ill.), according to the manufacturer's protocol. The Western blotting images were acquired using the Chemidoc-lt imaging system (UVP) and band intensity was calculated using imageJ software using “Gels and Plot lanes” plug-in.
Cell immunofluorescence: Chondrocytes were fixed for 10 min in 4% PFA in PBS and permeabilized for 30 min in 0.05% (w/v) saponin, 0.5% (w/v) BSA, 50 mM NH4C1 and 0.02% NaN3 in PBS (blocking buffer). The cells were incubated for 1 h with the primary antibodies, washed three times in PBS, incubated for 1 h with the secondary (Alexa fluor-labeled) antibody, washed three times in PBS, incubated for 20 min with 1 μg/ml Hoechst 33342 and finally mounted in Mowiol. All confocal experiments showing colocalization were acquired using slice thickness of 0.5 mm using the LSM 710 confocal microscope equipped with a 63×1.4 numerical aperture oil objective. Colocalization was measured using imageJ software using “JACoP” plug-in.
Results are given as means±standard errors of the means. Statistical analyses is performed using an unpaired, two-tailed Student t test. For all experiments significance is be indicated as follows: *, P≤0.05; **, P≤0.01; ***, P≤0.001.
The data provided by the inventors show a previously unanticipated role of chondrocyte autophagy in bone growth. Without being bound to theory, during early post-natal skeletogenesis, FGF18-FGFR4 complex induces the activation of INK kinase, which phosphorylates Bcl2 leading to the disruption of the Bcl2-Beclin 1 interaction and to the activation of the Beclin 1/Vps34 complex. This process leads to the production of a pool of PI3P required for autophagosome (AV) formation in chondrocytes. The induction of autophagy maintains PC2 homeostasis and prevents accumulation of PC2 in the ER during phases of high PC2 secretion. Chondrocyte autophagy appears to be dispensable when low levels of PC2 secretion are needed (e.g. pre-natal bone growth).Chondrocyte autophagy maintains the balance between synthesis, folding and secretion of PC2 in the ER during bone growth. This role is particularly important when PC2 synthesis is increased and massive secretion is needed to satisfy the high demand during post-natal bone growth. In these conditions a fraction of newly synthesized PC2 is degraded through autophagy probably due to imperfect folding or assembly.
Without being bound to theory, FGFR4 may regulate bone growth, at least in part; this occurs through modulation of autophagy.
Disruption of autophagy may lead to reduced femoral and tibial length (mainly post-natal role) and to deficient Col2 deposition in ECM (post-natal role); defective FGF signaling leads to defects in Col2 deposition in the ECM. Further pathogenetic mechanism can occur, leading to defects in the bone growth.
The inventors have demonstrated for the first time that activation of Beclin 1/Vps34 complex is beneficial in pathologies associated with bones developmental dysfunction, in particular long bones. Molecules according to the present invention are moreover capable of rescuing Col2 deposition defects and bone growth defects associated with bone growth disorders.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/073149 | 9/28/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62233687 | Sep 2015 | US |
