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WO2021011930A1 - Protéines de fusion slirp et leur utilisation pour le traitement du syndrome de leigh - Google Patents

Protéines de fusion slirp et leur utilisation pour le traitement du syndrome de leigh Download PDF

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Publication number
WO2021011930A1
WO2021011930A1 PCT/US2020/042685 US2020042685W WO2021011930A1 WO 2021011930 A1 WO2021011930 A1 WO 2021011930A1 US 2020042685 W US2020042685 W US 2020042685W WO 2021011930 A1 WO2021011930 A1 WO 2021011930A1
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slirp
subject
cell
seq
fusion protein
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Joan David BETTOUN
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Larimar Therapeutics Inc
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Chondrial Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)

Definitions

  • Leigh Syndrome is a genetic disorder associated with mutations in one of numerous nuclear genes or genes encoded by mitochondrial DNA. Genetic defects associated with Leigh Syndrome may include mutations in genes encoding OXPHOS enzymes and their assembly factors, or may be associated with defects in mitochondrial DNA maintenance, gene expression and protein synthesis; cofactor biosynthesis (lipoic acid and coenzyme Q10); mitochondrial membrane lipid remodeling; pyruvate dehydrogenase; biotinidase; and vitamin transporters.
  • Leigh syndrome is characterized by decompensation (often with elevated lactate levels in blood and/or CSF) during an intercurrent illness. It is typically associated with psychomotor retardation or regression, often followed by transient or prolonged stabilization or even improvement, but inevitably resulting in eventual progressive neurologic decline, typically occurring in stepwise decrements. Neurologic manifestations include hypotonia, spasticity, movement disorders (including chorea), cerebellar ataxia, and peripheral neuropathy.
  • Extraneurologic manifestations may include hypertrophic cardiomyopathy, hypertrichosis, anemia, renal tubulopathy, liver involvement, ptosis, and muscle weakness. Onset is typically between ages three and 12 months; about 50% of affected individuals die by age three years, most often as a result of respiratory or cardiac failure. Later onset (including in adulthood) and long-term survival may occasionally occur.
  • Leigh Syndrome French Canadian type LSFC
  • LRPPRC Leucine Rich Pentatricopeptide Repeat Containing
  • LSFC has a clinical onset at the age of between 0-8 months, and affected children present neurological disorder, mild regression of psychomotor skills, hypotonia, congenital cardiomyopathy and other congenital malformations, cognitive and speech impairment, energy deficit and deadly lactic acidosis episodes, leading to death between 3 and 10 years of age. Death results from related health problems (seizures, neurological/metabolic crises).
  • LSFC was originally reported in patients from the Saguenay-Lac St-Jean region of Quebec, with an incidence of 1 in 2,000 affected births in that region, while the general incidence outside of Quebec is 1 in 40,000.
  • the general hallmark of the intracellular phenotype for Leigh Syndrome is characterized by a cytochrome C oxidase (COX) deficiency, leading to congenital chronic lactic acidosis, detectable in serum and cerebro-spinal fluid (CSF).
  • COX cytochrome C oxidase
  • CSF cerebro-spinal fluid
  • SLIRP SRA stem-loop-interacting RNA-binding protein
  • RRM RNA-recognition motif
  • Protein replacement or supplementation therapy is a well-established approach to metabolic diseases, such as diabetes, lysosomal storage disorders and hemophilia.
  • the present disclosure relates to a novel treatment for Leigh Syndromes, e.g ., LSFC, by providing excess SLIRP protein.
  • novel SLIRP fusion proteins are provided in the present disclosure, where SLIRP is fused to domains that facilitate its intracellular delivery.
  • the SLIRP fusion proteins may be used as therapeutics for the treatment of Leigh Syndrome, e.g., Leigh Syndrome French Canadian type (LSFC).
  • an aspect of the disclosure relates to providing a SLIRP fusion protein or any variants or derivatives thereof, and at least one additional peptide, such as a cell penetrating peptide (CPP) or any variants or derivatives thereof.
  • CPP cell penetrating peptide
  • nucleotide sequences encoding the SLIRP fusion proteins provided in the disclosure and vectors comprising thereof for intracellular production of the fusion proteins.
  • Embodiments of the disclosure further provide a conjugate, a pharmaceutical composition, a therapeutic compound and a cell comprising the SLIRP fusion protein.
  • the present disclosure provides a method of treating Leigh Syndrome, that comprises administering to a subject in need thereof an effective amount of a SLIRP compound, such that the Leigh Syndrome in the subject is treated.
  • the Leigh Syndrome is Leigh Syndrome, French Canadian Type (LSFC).
  • the present disclosure also provides a method of treating LSFC, the method comprising administering to a subject in need thereof an effective amount of a SLIRP therapeutic compound, such that the LSFC in the subject is treated.
  • administering of the SLIRP therapeutic compound treats lactic acidosis in the subject.
  • the lactic acidosis is characterized by increased levels of lactic acid in at least one bodily fluid in the subject.
  • the bodily fluid is selected from the group consisting of blood, cerebrospinal fluid (CSF) and urine.
  • the present disclosure also provides a method of modulating LRPPRC in a subject affected by LSFC that comprises administering to the subject an effective amount of a SLIRP therapeutic compound, such that the LRPPRC protein in the subject is modulated.
  • a SLIRP therapeutic compound such that the LRPPRC protein in the subject is modulated.
  • the transcript level of at least one downstream target of LRPPRC is modulated is the subject.
  • mitochondrial RNA is stabilized in the the subject.
  • the present disclosure also provides a method of modulating LRPPRC in a cell that comprises contacting the cell with an effective amount of a SLIRP therapeutic compound, such that the LRPPRC in the cell is modulated.
  • transcript level of at least one downstream target of LRPPRC is modulated is the cell.
  • the cell is in a subject, e.g ., a subject is affected by LSFC.
  • the present invention also provides a method of treating lactic acidosis, the method comprising administering to a subject in need thereof an effective amount of a SLIRP therapeutic compound, such that the lactic acidosis in the subject is treated.
  • the lactic acidosis is characterized by increased levels of lactic acid in at least one bodily fluid in the subject.
  • the at least one bodily fluid is selected from the group consisting of blood, cerebrospinal fluid (CSF) and urine.
  • the subject has Leigh Syndrome.
  • the subject has LSFC.
  • the SLIRP therapeutic compound comprises, of consists of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the SLIRP therapeutic compound comprises a fusion protein comprising an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the fusion protein further comprises a cell penetrating peptide (CPP).
  • CPP may comprise, or may consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to any of SEQ ID NOS. 3-12.
  • the CPP may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3.
  • the present disclosure also provides a fusion protein comprising, or consisting of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and a CPP.
  • the CPP may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to any of SEQ ID NOS. 3-12.
  • the CPP may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3.
  • the present disclosure also provides a nucleic acid encoding a fusion protein as described herein.
  • the present disclosure also provides an expression vector for introducing a fusion protein into a cell that comprises the nucleic acid as described herein.
  • the expression vector is selected from the group consisting of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, a phagemid, a baculovirus and a
  • the present disclosure also provides a cell comprising the fusion protein, the nucleic acid sequence or the expression vector as described herein.
  • the cell may be selected from the group consisting of a muscle progenitor cell, a neuron progenitor cell, a bone marrow stem cell, a bacterial cell line and an yeast cell line.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein as described herein and a pharmaceutically acceptable diluent, carrier, additive or excipient.
  • Figs, la-lb present schematic designs of exemplary SLIRP fusion proteins; in accordance with embodiments of the disclosure.
  • Fig. la shows a schematic of a SLIRP fusion protein comprising TAT fused to the N-terminus of SLIRP; in accordance with an embodiment of the disclosure.
  • Fig. lb shows a schematic of a SLIRP fusion protein comprising TAT fused to the C-terminus of SLIRP; in accordance with an embodiment of the disclosure.
  • Figs. 2a-2b show the results of cells transfected with either shRN A- SCRAMBLE (Scramble 5-Clone) or shRNA-LRPPRC (Clones KD-1C and KD-2C).
  • Fig. 2a is a photo of a representative Western blot showing LRPPRC and mitochondrial Cytochrome Oxidase II (mt- C02) protein levels, and b-actin as an internal control, in cells transfected with Scramble 5- Clone (Scr-5), or with shRNA-LRPPRC (two clones, KD-1C and KD-2C), in accordance with an embodiment of the disclosure.
  • 2b is a graph showing the percentage expression level of LRPPRC protein and mtC02 in the cells transfected with Scr-5, or with shRNA-LRPPRC (KD- 1C and KD-2C), in accordance with an embodiment of the disclosure.
  • Figs. 3a-3b are a schematic of a mammalian (Fig. 3a) and a bacterial (Fig. 3b) expression vector, used for expression of the SLIRP fusion proteins, in accordance with an embodiment of the disclosure.
  • the present disclosure provides methods of treating Leigh Syndrome, such as Leigh Syndrome, French Canadian Type (LSFC), that involve SLIRP supplementation therapy.
  • the methods of the present disclosure comprise administering to a subject in need thereof an effective amount of a SLIRP therapeutic compound, such that the Leigh Syndrome in the subject is treated.
  • SLIRP supplementation therapy refers to supplementation of SLIRP in a subject, which results in increased levels, expression and/or activity of SLIRP in the subject.
  • SLIRP supplementation therapy involves the administration of a SLIRP therapeutic compound to a subject in need thereof.
  • SLIRP therapeutic compound which is used interchangeably herein with the term“SLIRP compound”, refers to any polypeptide that comprises, or any nucleic acid construct that is capable of providing a SLIRP protein.
  • SLIRP SRA stem-loop-interacting RNA-binding protein
  • RRM RNA-recognition motif
  • SLIRP is found ubiquitously in all tissues and with highest level in heart, liver, skeletal muscle and testis.
  • the terms“SLIRP protein” and“SLIRP polypeptide”, used interchangeably herein, refer to SLIRP from a human or a non-human subject, or a variant, fragment or derivative of SLIRP (e.g, biologically active fragment of SLIRP).
  • a“SLIRP protein” is a human SLIRP protein, or a variant, fragment or derivative thereof.
  • a SLIRP polypeptide may be the full-length, 109 amino acid (aa)-long SLIRP polypeptide, e.g, the human full-length SLIRP polypeptide (SEQ ID NO: 1), or a functional analogue, derivative or fragment thereof.
  • the SLIRP polypeptide may be the SLIRP RNA recognition motif (RRM), an 84 aa-long peptide, e.g, a human SLIRP RRM (SEQ ID NO: 2) or a functional analogue or derivative thereof.
  • a SLIRP polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO: 2, as shown in Table 1.
  • a SLIRP polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • a SLIRP therapeutic compound of the disclosure may encompass a SLIRP polypeptide, including e.g., SEQ ID NO: 1 or SEQ ID NO: 2.
  • the term“SLIRP therapeutic compound” or“SLIRP compound”, as used herein, also comprises a SLIRP fusion protein.
  • the term“SLIRP fusion protein” refers to a protein comprising SLIRP, or a variant, fragment or derivative of SLIRP (e.g, a biologically active variant, fragment or derivative of SLIRP), fused to at least one different amino acid sequence, e.g, a full length protein or a fragment of a different protein, or to a peptide.
  • the SLIRP fusion protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 fused to at least one different amino acid sequence, e.g., a full length protein or a fragment of a different protein, or to a peptide.
  • the SLIRP fusion protein comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 fused to at least one different amino acid sequence, e.g., a full length protein or a fragment of a different protein, or to a peptide.
  • the SLIRP fusion protein comprises SLIRP, or a variant, fragment (e.g, a biologically active fragment) or derivative thereof, fused to a cell penetrating peptide (CPP) as described herein.
  • CPP cell penetrating peptide
  • the CPP is the transduction domain of HIV-TAT (e.g, SEQ ID NO: 3).
  • the at least one different amino acid sequence comprised in a fusion protein of the present disclosure may comprise a cell penetrating peptide (CPP).
  • CPPs cell penetrating peptides
  • a CPP may facilitate the delivery of the fusion protein to a cell, e.g ., a recipient cell.
  • a CPP useful in the context of the present invention may be any CPP known to a person skilled in the art.
  • a CPP comprised in a fusion protein of the present disclosure may be any CPP listed in the Database of Cell-Penetrating Peptides CPPsite 2.0, the entire contents of which are hereby incorporated herein by reference.
  • a CPP useful in the context of the present invention may comprise an aromatic cationic peptide as described, e.g. , in US 10,576,124, the entire contents of which are hereby incorporated herein by reference.
  • an aromatic cationic peptide may be 2', 6' dimethyl-Tyr-D-Arg-Phe- Lys-NFh.
  • a CPP useful in the context of the present invention may be selected from the group consisting of HIV-TAT, the transduction domain of HIV-TAT (which may also be referred to herein as“TAT”), galanin, mastoparan, transportan, penetratin, polyarginine, or VP22.
  • TAT the transduction domain of HIV-TAT
  • galanin the transduction domain of HIV-TAT
  • mastoparan the transduction domain of HIV-TAT
  • transportan e.g., penetratin, polyarginine
  • VP22 VP22
  • Exemplary CPPs that may be used in a SLIRP fusion protein are shown in Table 2 below. Table 2.
  • the CPP comprised in a fusion protein useful in the context of the present disclosure may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to any one of SEQ ID NOs: 3-12 as listed in Table 2.
  • the CPP may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3.
  • the CPP may comprise, or consist of, an amino acid sequence with at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 4.
  • the CPP may comprise an amino acid sequence of any one of SEQ ID NOs: 3-12, e.g ., SEQ ID NO: 3. In some embodiments, the CPP comprises or consists of the amino acid sequence of SEQ ID NO: 4.
  • a SLIRP fusion protein may comprise SLIRP, or a variant, fragment or derivative thereof, and CPP, wherein the CPP is fused to the SLIRP at the N- terminus of the SLIRP.
  • a SLIRP fusion protein may comprise SLIRP, or a variant, fragment or derivative thereof, and CPP, wherein the CPP is fused to SLIRP at the C- terminus of the SLIRP.
  • the CPP may be fused to the SLIRP directly, or indirectly, e.g. , through a linker).
  • the term“directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.
  • the last amino acid of the C-terminal end of said polypeptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said polypeptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.
  • linker refers to a sequence of at least one amino acid that links the polypeptides and proteins comprised in the SLIRP fusion protein utilized in the present disclosure. Such a linker may be useful to prevent steric hindrances.
  • the length of a linker may vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners of the fusion protein.
  • Linkers have been described in the literature both as endogenous, naturally occurring, playing a role in separating domains within a protein or for the formation of dimers. Alternatively, linkers have been described in recombinant technology for the generation of fusion proteins.
  • the term“SLIRP therapeutic compound” or“SLIRP compound”, as used herein, also encompasses a nucleic acid sequence, e.g ., a vector, encoding a SLIRP polypeptide or a variant, fragment or derivative thereof.
  • the term“SLIRP therapeutic compound” or“SLIRP compound”, as used herein encompasses a nucleic acid sequence, e.g. , a vector, encoding an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, fragment of derivative thereof.
  • the nucleic acid sequence may be in a cell, e.g.
  • a SLIRP polypeptide e.g., hSLIRP
  • a SLIRP fusion protein e.g., comprising SLIRP (e.g., hSLIRP).
  • the term“SLIRP therapeutic compound” or“SLIRP compound”, comprises a polypeptide comprising, or consisting of, a SLIRP fragment.
  • the“SLIRP therapeutic compound” or“SLIRP compound” comprises a nucleic acid sequence encoding a polypeptide comprising, or consisting of, a SLIRP fragment.
  • the term“SLIRP fragment”, used interchangeably herein with the terms“fragment of SLIRP” and“a portion of SLIRP”, encompasses any polypeptide that is shorter than a full-length SLIRP, e.g. , shorter than human full-length SLIRP (SEQ ID NO: 1) or SLIRP RRM (e.g, SEQ ID NO: 2).
  • a SLIRP fragment may have a biological activity of SLIRP, i.e., a biologically active fragment of SLIRP.
  • a SLIRP fragment may have substantially the same biological activity as full length SLIRP, e.g, human full-length SLIRP (SEQ ID NO: 1).
  • a SLIRP fragment may have partial biological activity of SLIRP, for example, about 30% to about 100%, e.g., about 30% to about 60%, about 50% to about 80% or about 60% to about 95% of the biological activity of full length SLIRP, e.g., human full-length SLIRP (SEQ ID NO; 1).
  • SLIRP may be administered through delivery of a SLIRP polypeptide or through delivery of a nucleic acid encoding a SLIRP polypeptide.
  • SLIRP may be full length SLIRP, a SLIRP fragment, a SLIRP fusion protein, or a variant, fragment or derivative thereof.
  • a SLIRP fusion protein may comprise a SLIRP fragment or full length SLIRP, fused to a different protein or to a fragment thereof, or to a peptide.
  • the present disclosure also provides a SLIRP fusion protein that is useful as a SLIRP therapeutic compound for the treatment of Leigh Syndrome, e.g ., LSFC.
  • the SLIRP fusion protein provided herein may comprise a SLIRP polypeptide (e.g., a hSLIRP, e.g., SEQ ID NO: 1, or SEQ ID NO:2) fused to a domain that facilitates its intracellular delivery, e.g. , CPP.
  • a SLIRP fusion protein comprises a SLIRP polypeptide and a cell penetrating peptide (CPP).
  • a SLIRP polypeptide may be the full-length, 109 amino acid (aa)-long SLIRP polypeptide (e.g, SEQ ID NO: 1), or any functional variant, analogue, derivative or fragment thereof.
  • the SLIRP polypeptide may be the SLIRP RNA recognition motif (RRM), an 84 aa-long peptide (SEQ ID NO: 2) or any functional variant, analogue, derivative or fragment thereof.
  • the SLIRP fusion protein may also comprise a CPP domain, e.g., any of SEQ ID NOS. 3-10, or an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NOS. 3-10.
  • the SLIRP fusion protein comprises the transduction domain of HIV-TAT having an amino acid sequence YGRKKRRQRRR (SEQ ID NO: 3).
  • a CPP may have a sequence as described herein, or a sequence having at least 85%, or at least 90% or at least 95% homology with these sequences.
  • homologous sequences may be found in one or more of the following databases: GenBank, Protein Data Bank (PDB), UniProt Knowledgebase (UniProtKB), SwissProt, Protein Information Resource (PIR), or Protein Research Foundation (PRF).
  • “derivative”, as used herein, refers to amino acid sequences (polypeptides), which differ from the polypeptides specifically defined in the present disclosure by insertions, deletions, substitutions and modifications of amino acid residues that do not alter the activity of the original polypeptides.
  • “insertion/s”,“deletion/s” or“substitution/s”, as used herein it is meant any addition, deletion or replacement, respectively, of amino acid residues to the polypeptides of between 1 to 50 amino acid residues, or between 1 to 10 amino acid residues. More particularly, insertion/s, deletion/s or substitution/s may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. It should be noted that the insertion/s, deletion/s or substitution/s may occur in any position of the modified peptide, as well as in any of the N’ or C’ termini thereof.
  • derivative it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present disclosure.
  • a derivative, a variant and an analogue of any one of the proteins or the peptides comprised in the fusion protein provided in the present disclosure, e.g ., SLIRP, will have the same or similar biological activity as its native form.
  • the SLIRP polypeptide sequence and any polynucleotide encoding the SLIRP polypeptide sequence applies to SLIRP orthologs or homologues having a sequence homology or identity to the SLIRP protein used as described herein after, of at least 50%, at least 60%, or at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically as compared to the entire sequence of the SLIRP as denoted by SEQ ID NO. 1 or SEQ ID NO: 2.
  • homologs that comprise or consist of an amino acid sequence having a sequence identity of at least 50%, at least 60%, or at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher to SEQ ID NO. 1 or SEQ ID NO: 2, may be comprised in the SLIRP therapeutic compound, e.g. , SLIRP fusion protein, described herein.
  • the present disclosure also provides a nucleic acid encoding a SLIRP fusion protein as defined in the present disclosure, and any variants thereof.
  • a nucleic acid encoding a SLIRP fusion protein described herein includes any sequence having homology of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the sequences encoding the fusion proteins provided herein.
  • nucleic acids is interchangeable with the term“polynucleotide(s)” and it generally refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or any combination thereof.
  • Nucleic acids include, without limitation, single- and double-stranded nucleic acids.
  • variants referred to in this disclosure optionally include conservatively substituted variants that apply to both amino acid and nucleic acid sequences.
  • conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are silent variations, which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • conservative substitution of amino acid sequences one of skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following groups each contain amino acids that are conservative substitutions for one another:
  • the term“identical” or“percent identity” in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence.
  • the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program
  • the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10.
  • the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule.
  • the term "being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.
  • the present invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the h erein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s).
  • a SLIRP therapeutic compound may be administered by delivery of a SLIRP polypeptide (e.g ., a fusion protein comprising a SLIRP polypeptide) or through delivery of a nucleic acid encoding a SLIRP polypeptide (e.g., a fusion protein comprising a SLIRP polypeptide).
  • a SLIRP polypeptide e.g ., a fusion protein comprising a SLIRP polypeptide
  • a nucleic acid encoding a SLIRP polypeptide e.g., a fusion protein comprising a SLIRP polypeptide
  • the present disclosure also relates to providing an expression vector for producing a SLIRP polypeptide or SLIRP fusion protein as defined herein in a cell (mammalian, bacterial or fungal).
  • a SLIRP therapeutic compound may be administered in accordance with methods of the present invention as an expression vector for producing a SLIRP polypeptide or a SLIRP fusion protein (such as a mammalian, bacterial or fungal cell).
  • the expression vector may comprise a nucleic acid encoding a SLIRP polypeptide or a SLIRP fusion protein as defined herein.
  • the expression vector may be a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, a phagemid, a baculovirus, or any combination thereof.
  • Vectors may enable the integration of DNA fragments or nucleic acid sequences into the genome of the host or enable expression of genetic elements that are not integrated.
  • Vectors are typically self-replicating DNA or RNA constructs containing the desired nucleic acid sequences, and operably linked genetic control elements that are recognized in a suitable host cell and effect the translation of the desired spacers.
  • the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such system typically includes a transcriptional promoter and transcription enhancers to elevate the level of RNA expression. These are usually referred to as“expression vectors”.
  • Vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • control and regulatory elements may include promoters, terminators and other expression control elements.
  • Such regulatory elements are described in the art and known to the skilled artisan.
  • any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding any desired SLIRP proteins or SLIRP fusion protein as described herein.
  • the phrase“operatively-linked” is intended to mean attached in a manner which allows for transgene transcription.
  • encoding is intended to mean that the subject nucleic acid may be transcribed and translated into either the desired SLIRP polypeptide or the subject protein in an appropriate expression system, e.g., when the subject nucleic acid is linked to appropriate control sequences such as promoter and enhancer elements in a suitable vector (e.g., an expression vector) and when the vector is introduced into an appropriate system or cell.
  • a suitable vector e.g., an expression vector
  • a vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector-containing cells.
  • Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function, and which are, or become, known in the art are suitable for use herein.
  • a vector may also be referred to as a construct, or as recombinant nucleic acid.
  • the term“recombinant DNA”,“recombinant nucleic acid sequence” or “recombinant gene” refers to a nucleic acid comprising an open reading frame (ORF) encoding a SLIRP fusion protein as described herein.
  • a SLIRP therapeutic compound comprising a nucleic acid encoding a SLIRP protein or SLIRP fusion protein, e.g., comprising SLIRP or a variant, fragment or derivative thereof, may also be delivered in accordance with methods of the present disclosure by viral gene
  • a SLIRP therapeutic compound comprising a nucleic acid encoding a SLIRP protein or SLIRP fusion protein, e.g., comprising SLIRP or a variant, fragment or derivative thereof, may also be delivered in accordance with methods of the present disclosure using a non-viral delivery system.
  • Non-limiting examples of non-viral delivery systems include, e.g, lipid-based DNA vectors, and polymeric DNA vectors, as described in Yin et al. , Nature Reviews Genetics 2014, 15, 541-555, the entire contents of which are incorporated herein by reference.
  • a cell comprising any one of a SLIRP protein, e.g, a SLIRP fusion protein, a nucleic acid encoding thereof, a vector comprising said nucleic acid, or any combination thereof, as defined in the disclosure.
  • a cell may be any one of a muscle or muscle progenitor cell, a fibroblast, an epidermal cell, a cardiac cell, a stem cell, an embryonic stem cell, a pluripotent cell, a neuron, a bone marrow stem cell, and including yeast or bacteria cell lines.
  • a SLIRP therapeutic compound may be administered as a part of a pharmaceutical composition comprising a SLIRP therapeutic compound and a pharmaceutically acceptable diluent, carrier, additive and/or excipient.
  • a pharmaceutical composition comprising a SLIRP therapeutic compound, e.g. , a SLIRP fusion protein, as defined in the disclosure; and a pharmaceutically acceptable diluent, carrier, additive and/or excipient.
  • a pharmaceutical composition may be delivered to cells in vitro , for example by contacting the cells with the pharmaceutical composition.
  • the pharmaceutical composition provided in the present disclosure may be administered orally, parenterally, by inhalation spray, rectally, intradermally, transdermally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices.
  • parenteral includes subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques.
  • Injectable pharmaceutical compositions for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable pharmaceutical composition may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are useful in the preparation of injectables.
  • Dimethyl acetamide, surfactants including ionic and non-ionic detergents, and polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.
  • Suppositories for rectal administration of the fusion proteins discussed herein can be prepared by mixing the active agent with a suitable non-irritating excipient such as cocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, or polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature, and which will therefore melt in the rectum and release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, or polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature, and which will therefore melt in the rectum and release the drug.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • the fusion proteins or compositions of this disclosure are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration.
  • a SLIRP therapeutic compound or a composition comprising a SLIRP therapeutic compound can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.
  • Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.
  • the dosage forms can also comprise buffering agents such as sodium citrate, or magnesium or calcium carbonate or bicarbonate. Tablets and pills can additionally be prepared with enteric coatings.
  • compositions for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration.
  • the SLIRP therapeutic compounds, e.g., SLIRP fusion proteins, or compositions can be dissolved in water,
  • polyethylene glycol polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.
  • Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.
  • Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
  • Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the patient and the particular mode of administration.
  • a SLIRP therapeutic compound e.g. , SLIRP polypeptide, or a SLIRP fusion protein
  • a protein delivery system may be prepared as part of a protein delivery system.
  • a broad overview of protein delivery systems may be found, for example, in Banga, A. K. (2015) Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd ed., CRC Press, Taylor & Francis Group, the entire content of which is incorporated by reference herein.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules may contain the SLIRP therapeutic compound, such as SLIRP polypeptide or SLIRP fusion protein, as a central core. In microspheres, the SLIRP therapeutic compound may be dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J.
  • a SLIRP therapeutic compound e.g., a SLIRP fusion protein
  • a subject in need thereof in a therapeutically effective amount.
  • Drug dosages and regimens for treating various conditions are well known in the art. Note in this regard, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 1996, Ninth Edition, McGraw-Hill, New York.
  • the SLIRP therapeutic compounds, e.g, SLIRP fusion proteins of the present disclosure can be administered by a variety of methods, including, for example, orally, enterally, mucosally, percutaneously, or parenterally.
  • parenteral administration is preferred, especially by intravenous, intramuscular, subcutaneous, intracutaneous, intraarticular, intrathecal, and intraperitoneal infusion or injection, including continuous infusions or intermittent infusions with pumps available to those skilled in the art.
  • the fusion protein may be administered by means of micro-encapsulated preparations, for example based on liposomes.
  • treatment of a subject affected by Leigh Syndrome e.g., LSFC
  • LSFC Leigh Syndrome
  • Upregulation of the endogenous SLIRP gene may be triggered through a compound, a drug, or any agent that affects the mitochondrial pathway.
  • the present disclosure provides methods for treating Leigh Syndrome, e.g, LSFC, that comprise administering to a subject in need thereof an effective amount of a SLIRP therapeutic compound, e.g, SLIRP fusion protein, as described herein.
  • a SLIRP therapeutic compound e.g, SLIRP fusion protein
  • Leigh Syndrome encompasses any Leigh Syndrome known in the art.
  • Leigh Syndrome is a genetic neurological disorder that may be associated with dysfunction in proteins encoded by nuclear genes or mitochondrial genes and may be inherited in an autosomal recessive manner, X-linked manner or mitochondrial (maternal) manner.
  • Leigh Syndrome may be diagnosed based on the following diagnostic criteria: a) presence of characteristic clinical presentation as described herein; b) presence of lesions in the basal ganglia, cerebellum and brainstem that are identifiable by brain MRI; c) lactic acidosis, characterized by elevated lactate levels in bodily fluids, such as blood, cerebrospinal fluid (CSF) and/or urine; and d) presence of a pathogenic variant of one of the genes associated with Leigh Syndrome, as described herein.
  • diagnostic criteria a) presence of characteristic clinical presentation as described herein; b) presence of lesions in the basal ganglia, cerebellum and brainstem that are identifiable by brain MRI; c) lactic acidosis, characterized by elevated lactate levels in bodily fluids, such as blood, cerebrospinal fluid (CSF) and/or urine; and d) presence of a pathogenic variant of one of the genes associated with Leigh Syndrome, as described herein.
  • CSF cerebrospinal fluid
  • the term“Leigh Syndrome”, as used herein, also encompasses “Leigh-like Syndromes”, i.e., disorders with clinical and other features that are strongly suggestive of Leigh Syndrome but that do not fulfill the stringent diagnostic criteria for Leigh Syndrome because, e.g ., of absence of at least one of the diagnostic criteria as described above or presence of at least one feature that does not fit the diagnostic criteria for Leigh Syndrome as described above.
  • Leigh Syndrome is characterized by progressive loss of mental and movement abilities (psychomotor regression).
  • the characteristic clinical presentation of Leigh Syndrome that may be used for diagnosis includes neurologic manifestations, such as weak muscle tone (hypotonia), spasticity, movement disorders (including chorea), ataxia, cerebellar ataxia, and peripheral neuropathy.
  • the characteristic clinical presentation of Leigh Syndrome may also include extraneurologic manifestations, such as hypertrophic cardiomyopathy, hypertrichosis, anemia, renal tubulopathy, liver involvement, ptosis, and muscle weakness.
  • Leigh Syndrome may be associated with mutations in numerous genes, including nuclear and mitochondrial genes as described in, e.g. , Rahman S, Thorburn D. Nuclear Gene-Encoded Leigh Syndrome Spectrum Overview. 2015 Oct 1 [Updated 2020 Jul 16] In: Adam MP, Ardinger HH, Pagon RA, et al. , editors. GeneReviews® [Internet] Seattle (WA): University of Washington, Seattle; 1993-2020 and Thorburn DR, Rahman J, Rahman S. Mitochondrial DNA- Associated Leigh Syndrome and NARP. 2003 Oct 30 [Updated 2017 Sep 28] In: Adam MP, Ardinger HH, Pagon RA, et al., editors.
  • Leigh Syndrome within the context of the present disclosure may be associated with a mutation in a nuclear gene, wherein the nuclear gene is selected from the group consisting of: NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8,
  • the Leigh Syndrome may be LSFC, which is associated with mutations in the LRPPRC gene.
  • the Leigh Syndrome may be associated with a mutation in a gene encoded by mitochondrial DNA, wherein the gene is selected from the group consisting of: MT- ATP6 , MT-ND3 , MT-ND5 , MT-ND6, MT-C03 , MT-ND1, MT-ND2, MT-ND4, MT-TI , MT-TK , MT-TL1 , MT-TL2 , MT-TV and MT-TW.
  • treating Leigh Syndrome encompasses ameliorating, improving or achieving a reduction in the severity of Leigh Syndrome, such as LSFC, e.g ., ameliorating, improving or achieving a reduction in at least one symptom or indicator associated with Leigh Syndrome in a subject.
  • Treatment Leigh Syndrome also refers to ameliorating, improving or achieving a reduction in the severity of Leigh Syndrome, such as LSFC, e.g ., ameliorating, improving or achieving a reduction in at least one symptom or indicator associated with Leigh Syndrome in a subject.
  • the at least one symptom or indicator associated with Leigh Syndrome may be selected from the group consisting of developmental delay, ataxia, hypotonia, spasticity, movement disorders, cerebellar ataxia, peripheral neuropathy, cardiomyopathy, hypertrichosis, anemia, renal tubulopathy, ptosis, muscle weakness, brain lesions and lactic acidosis.
  • the term“treating Leigh Syndrome”, as used herein, encompasses ameliorating, improving of achieving a reduction in lactic acidosis in a subject.
  • the lactic acidosis is characterized by increased lactic acid levels in at least one bodily fluid, such as blood, CSF and/or urine, in a subject.
  • the Leigh Syndrome may be LSFC.
  • the present disclosure also provides methods for treating lactic acidosis that comprise administering to a subject in need thereof a SLIRP therapeutic compound as described herein.
  • the lactic acidosis may be characterized by increased lactic acid levels in at least one bodily fluid, such as blood, CSF and/or urine, in a subject.
  • the subject may have Leigh Syndrome, e.g. , LSFC.
  • the term“treating lactic acidosis”, as used herein, encompasses ameliorating, improving or achieving a reduction in the severity of lactic acidosis, delaying progression of acidosis, or preventing an increase in the severity of acidosis in a subject.
  • “treating lactic acidosis”, as used herein, encompasses reducing levels of lactic acid in at least one bodily fluid in a subject, e.g ., blood, CSF and/or urine.
  • Levels of lactic acid, in at least one bodily fluid in a subject may be measured by any method known to one of skill in the art.
  • the term“effective amount” of a SLIRP therapeutic compound is that amount which is necessary or sufficient to treat Leigh Syndrome, e.g. , ameliorate, improve or achieve a reduction in the severity of at least one symptom or indicator associated with Leigh Syndrome, e.g. , LSFC.
  • the term“effective amount” of a SLIRP therapeutic compound is that amount which is necessary or sufficient to achieve a reduction in lactic acidosis in a subject, e.g. , to achieve a reduction in levels of lactic acid in at least one bodily fluid, such as blood, CSF and/or urine, in a subject.
  • One of ordinary skill in the art can determine an effective amount of a SLIRP therapeutic compound for administration to a subject. Drug dosages and regimens for treating various conditions are well known in the art. Note in this regard, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 1996, Ninth Edition, McGraw-Hill,
  • SLIRP therapeutic compound may be combined with carrier materials to produce a single dosage form will vary depending upon the patient and the particular mode of administration.
  • a method of treatment of Leigh Syndrome e.g. , LSFC
  • the method comprising administering a therapeutically effective amount of a SLIRP therapeutic compound, e.g. , a SLIRP fusion protein, a nucleic acid encoding said SLIRP fusion protein, a vector comprising said nucleic acid, a conjugate comprising a SLIRP protein, or a pharmaceutical composition as described herein, to a subject suffering from Leigh Syndrome, e.g. , LSFC.
  • the SLIRP therapeutic compound comprises a SLIRP protein.
  • the SLIRP therapeutic compound comprises a SLIRP fusion protein.
  • the SLIRP therapeutic compound comprises a nucleic acid encoding a SLIRP protein, e.g., a SLIRP fusion protein. In one embodiment, the SLIRP therapeutic compound comprises a vector comprising a nucleic acid encoding a SLIRP protein, e.g, a SLIRP fusion protein. In one embodiment, the SLIRP therapeutic compound comprises a conjugate comprising a SLIRP protein. In some embodiments, the present disclosure also provides a method of modulating the level of LRPPRC protein, or stabilizing LRPPRC in a cell, the method comprising administering an effective amount of a SLIRP fusion protein as described herein to a cell. This aspect may be particularly relevant to modulation or stabilization of LRPPRC variants which result from the expression of LRPPRC recessive alleles that are the cause of LSFC.
  • LRPPRC protein stability may be detected and measured through techniques well known in the art, including, for example, the analysis of LRPPRC protein half-life or LRPPRC turnover in a cell.
  • modulate LRPPRC refers to causing increased or decreased amounts of LRPPRC protein, in a subject, e.g ., a subject being administered a SLIRP compound, or a cell being contacted with a SLIRP compound.
  • modulate LRPPRC also refers to causing increased or decreased activity of the LRPPRC protein.
  • modulated level of a protein refers to a changed, e.g. , increased or decreased amount or activity of a particular protein, for example LRPPRC, or cytochrome c oxidase (COX) family members, such as COX2, as compared to the amount or activity of the protein in a control sample, when detected by any of the techniques known to the person skilled in the art.
  • a particular protein for example LRPPRC, or cytochrome c oxidase (COX) family members, such as COX2, as compared to the amount or activity of the protein in a control sample, when detected by any of the techniques known to the person skilled in the art.
  • COX cytochrome c oxidase
  • The“amount” of a protein, or a protein level in a subject or in a cell is“significantly” higher or lower than the normal amount of said protein, if the amount of the protein is greater or less, respectively, than the normal level (e.g, level in a healthy subject of a threshold level) by an amount greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount.
  • the amount of the protein in a subject or in a cell can be considered“significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the protein.
  • the amount of the protein in the subject or in a cell can be considered“significantly” higher or lower than the normal amount (e.g, amount in a healthy subject or a threshold amount) if the amount is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, higher or lower, respectively, than the normal amount of the protein.
  • modulated level of a protein may also refer to an increased or decreased level of activity of a protein, e.g. , LRPPRC protein or SLIRP protein, which may, or may not be caused by increased or decreased amounts of the protein.
  • proteins which have their levels modulated following administration of a SLIRP therapeutic compound may be detected and quantitated by any suitable method for detecting proteins or polypeptides known to the person skilled in the art.
  • proteins may be detected by a variety of immunodetection methods, such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay (MA), immunoradiometric assay, fluoroimmunoassay,
  • ELISA enzyme linked immunosorbent assay
  • MA radioimmunoassay
  • fluoroimmunoassay fluoroimmunoassay
  • proteins which have their levels modulated by a SLIRP therapeutic compound may be detected and quantitated using protein mass spectrometry methods and instrumentation.
  • Protein mass spectrometry which may be achieved for example by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization
  • MALDI massive lipid-containing lipoprotein
  • TOF time-of-flight
  • FT-ICR Fourier transform ion cyclotron resonance
  • the proteins can also be measured in complex mixtures of proteins and molecules that co-exist in a biological medium or sample, however fractionation of the sample may be required and is contemplated herein.
  • Two methods are widely used to fractionate proteins, or their peptide products from an enzymatic digestion, two-dimensional gel electrophoresis (2D Gel) and high-performance liquid chromatography (LC or HPLC). Any other suitable methods known in the art for fractionating protein mixtures are also contemplated herein.
  • a SLIRP therapeutic compound may comprise full-length SLIRP (e.g., SEQ ID NO: 1), a fragment of full-length SLIRP (e.g., SLIRP RRM, such as SEQ ID NO: 2), a SLIRP fusion protein, or any variant, fragment or derivative thereof.
  • “subject” or“patient” can mean either a human or non-human animal, preferably a mammal.
  • a human subject may be referred to as a patient.
  • adjectives such as“substantially” and“about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the disclosure are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended.
  • the word“or” in the description and claims is considered to be the inclusive“or” (having the meaning of and/or) rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.
  • the Table below presents the sequences of human SLIRP protein full length (SEQ ID NO: 1) and SLIRP RNA recognition motif (RRM), amino acid residues 19-103 (SEQ ID NO: 2), obtained from the UnitProtKB reference database, https ://www.umprot. org/uni prot/09GZT3 and the sequences of examples of CPP peptides, according to embodiments of the disclosure.
  • Example 2 SLIRP Fusion Protein Delivery Genetic constructs encoding various exemplary SLIRP fusion proteins were cloned into mammalian cell expression vectors, for example pCMV6-AC-mGFP (Fig. 3a), after excising the mGFP from the vector and inserting the SLIRP fusion construct in that position. All protein encoding sequences are codon optimized for mammalian (human) expression.
  • His-Tag is a polyhistidine motif of between two and 10 Histidines which are added to proteins in order to facilitate protein purification.
  • a protein having a His-tag is brought into contact with a carrier on which a metal ion, for example nickel (Ni), is immobilized under the condition of pH 8 or higher, the histidine residue chelates the metal ion and binds to the carrier.
  • a metal ion for example nickel (Ni)
  • Ni nickel
  • Mammalian expression vectors are transfected into a cell line, for example HEK293 cells. Transfection may be effected by any technique available to the person skilled in the art. Examples of transfection techniques are:
  • Microinjection - expression vectors can be injected into the cell
  • Lipofectamine transfection - plasmids can be mixed with positively charged and neutral lipids, and this mixture is internalized in the cells through phagocytosis or membrane fusion;
  • Calcium phosphate precipitation - plasmids can be mixed with calcium phosphate, forming a complex that precipitates and that can subsequently be employed to transfect susceptible cells;
  • Electroporation - a high-voltage pulse is applied to the cells to create pores in the external membrane, which facilitates the entry of the expression vectors into the cells.
  • Example 3 Generation of an in vitro Cell Model for Leigh Syndrome French Canadian (LSFC) type HEK293 cells were transfected with LRPPRC short hairpin (sh)RNA contained in lentiviral particles, resulting in targeted gene silencing of LRPPRC.
  • LSFC Long Term Evolution
  • LRPPRC short hairpin (sh)RNA contained in lentiviral particles, resulting in targeted gene silencing of LRPPRC.
  • a control cell line transfected with a scramble sequence (Scr-5) was generated in parallel.
  • Figs. 2a and 2b show the analysis of the cell lines generated, the control Scramble 5-Clone, and LRPPRC knock down (KD) clones KD-1C and KD-2C.
  • LRPPRC protein levels in clones KD-1C and KD-2C were reduced by about 70% when compared to the levels of LRPPRC protein in the Scramble-5 clone (see the Western blot in Fig. 2a and the graph in Fig. 2b, quantifying the protein levels).
  • cytochrome c oxidase subunit 2 (mtCOX2) were also detected in the Western blot (Fig. 2a) and quantified (Fig. 2b) and were shown to be reduced by about 75% in the KD clones. Levels of mtCOX2 and function are one of the hallmarks of Leigh Syndrome.
  • LRPPRC KD cells are transfected with expression vectors encoding SLIRP fusion proteins as described herein.
  • Expression of SLIRP in LRPPRC KD cells is expected to trigger increased expression of mtCOX2 gene (transcript levels can be verified by Northern blot and RT-PCR) and increased levels of mtCOX2 protein (protein levels can be verified by Western blot).
  • Increased mtRNA stability is expected to be evidenced by longer polyA tails in transcripts of selected mtRNA transcripts, which can be verified by sequencing.
  • LSFC patient fibroblasts are collected from affected patients.
  • Cells are cultured in the appropriate conditions.
  • Cells may be maintained in high glucose DMEM medium supplemented with 10% FBS.
  • FBS 10% FBS
  • the cells are now allowed to become stationary or to remain confluent for more than 24 hours.
  • Cells are transfected with an expression vector encoding a SLIRP fusion or a control vector.
  • a detailed description of primary cell cultures may be found in: R Ian Freshney et al., Culture of animal cells: a manual of basic technique and specialized applications , Wiley -Blackwell, 2016, the entire contents of which are incorporated herein by reference.
  • LRPPRC primary culture cells are examined for LRPPRC and COX2 levels by Western blot.
  • Levels of LRPPRC and mtCOX2 proteins are expected to be higher in LKPPRC KD cells transfected with SLIRP fusion proteins when compared to cells transfected with a control vector.
  • Levels of mtRNA are expected to be stabilized upon SLIRP overexpression, and mtRNA transcripts polyA tails are expected to be longer than in cells transfected with control. The results are predicted to demonstrate that SLIRP can act as a therapeutic compound for LSFC, and minimize its symptoms.
  • Cells from an LRPPRC patient are collected and transfected with any one of the SLIRP fusion variants. Cells are tested, by staining or Western blot, for the expression of SLIRP protein variants. Transfected cells are delivered back to the LRPPRC patient. Recovery of cytochrome C oxidase pathway function is expected to be observed in graft tissues.
  • each of the verbs,“comprise” “include” and“have”, and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

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Abstract

L'invention concerne des composés thérapeutiques SLIRP, comprenant des protéines de fusion comprenant un polypeptide SLIRP et un peptide de pénétration cellulaire (CPP). L'invention concerne également des procédés d'utilisation de composés thérapeutiques SLIRP, y compris une protéine de fusion SLIRP, pour le traitement du syndrome de Leigh, par exemple la forme franco-canadienne du syndrome de Leigh (LSFC).
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