This application is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/EP2012/076697 filed Dec. 21, 2012, which designates the U.S., and which claims benefit under one or more of 35 U.S.C. § 119(a)-119(d) of European Application No. 11194796.6, filed Dec. 21, 2011, the content of which is incorporated herein by reference in its entirety.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created Nov. 30, 2016, is named Revised_SL_048262-082020-US.txt and is 2,223,693 bytes in size.
The invention relates to variants of yeast NDI1 gene, proteins encoded by the variants, and the uses of the variant genes, transcribed RNA and proteins in the treatment of disease, especially neurodegenerative disease.
Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder affecting 1/25,000 people, predominantly males1. Loss of central vision results from the degeneration of the retinal ganglion cell (RGC) layer and optic nerve2. In over 95% of patients the genetic pathogenesis of LHON involves mutations in genes encoding components of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex3 (complex I), which is involved in transfer of electrons from NADH to ubiquinone (coenzyme Q). Complex I is composed of forty-six subunits, seven of which are encoded by the mitochondrial genome, ND1-6 and ND4L. Mutations in five of the mitochondrially encoded subunits of complex I, ND1, ND4, ND4L, ND5 and ND6, are associated with LHON. There is growing evidence that mitochondrial dysfunction may be involved in a wide range of neurodegenerative disorders such as Alzheimer disease (AD), Huntington disease and dominant optic atrophy as well as multifactorial diseases including dry and wet age related macular degeneration (AMD), diabetic retinopathies and glaucoma4. It is perhaps not surprising that a tissue such as retina, with the most significant energy requirements of any mammalian tissues, may be particularly vulnerable to mitochondrial dysfunction. However, it is notable that such a dependency on energy metabolism in principle may provide an opportunity for the development of therapeutic interventions for such high energy-dependent tissues where a shift in energy metabolism may potentially provide substantial beneficial effects. Complex I dysfunction results in an increase of reactive oxygen species (ROS) and a decreased energy supply6. In mitochondria, ATP synthesis is coupled to oxygen consumption by the proton electrochemical gradient established across the mitochondrial inner membrane in the process termed oxidative phosphorylation7 (OXPHOS). Mitochondrial complex I mutations leading to respiratory chain dysfunction are hence linked to reduced oxygen consumption; a reliable measure of overall mitochondrial activity.
Interestingly, many LHON mutations are not fully penetrant, it seems that the appearance of the pathological features of the disorder may be influenced by genetic and environmental modifiers. For example, it has been observed that the T14484C mutation in the ND6 subunit tends to be associated with a better clinical outcome and at times recovery in visual function8. Furthermore, there has been some suggestion that certain mitochondrial genetic backgrounds may render patients more or less susceptible to a variety of disorders including LHON and that this may be linked to variations in oxygen consumption, the efficiency of electron transport and ATP production9. For example, the G11778A and T14484C LHON mutations on a mitochondrial haplogroup J or K background have been associated with an increased risk of visual loss10. Nuclear modifier genes can influence LHON progression and severity, for example, an x-linked modifier locus has been reported11. Additionally, smoking has been suggested as one of the environmental factors which can influence disease penetrance12. In addition, the male prevalence (5:1) of LHON may at last in part be influenced by oestrogens13. An interplay between the primary mutation, modifying nuclear genes, the mtDNA genetic background and environmental factors may collaborate to determine overall risk of visual loss for a given LHON patient.
While significant progress has been made with regard to understanding the genetic pathogenesis of LHON, development of gene therapies for LHON has been impeded by the need to deliver therapies to the mitochondria of RGCs. In addition, intragenic heterogeneity has made development of therapies complex. Allotopic or nuclear expression of mitochondrial genes is being explored as a potential therapeutic avenue for some mitochondrial disorders including ND4-linked LHON, although modifications may be required to facilitate import of expressed proteins into mitochondria14,15,16. A nuclear complementation approach using NDI1 has been considered as a potential therapy for Parkinson disease (PD)17. Additionally, recombinant adenoassociated virus (AAV) serotype 5 delivery of NDI1 into the optic layer of the superior colliculus of the brain, has recently been shown to provide significant benefit in a chemically-induced rat model of LHON using functional and histological readouts18. Whereas this represents an exciting and innovative strategy making use of transkingdom gene therapy, the mode of delivery may not be readily translatable to human LHON patients.
It is an object of the invention to overcome at least one of the above-referenced problems.
The invention relates to variants of the yeast NDI1 gene of SEQ ID NO: 1 which are codon optimised to provide for improved expression in mammalian cells, and/or modified to encode an immune optimised functional variant of NDI1 protein. Codon optimisation involves replacing codons which are common to yeast cells and uncommon to mammalian cells with synonomous codons which are common to mammalian cells. These are known as “silent changes” as they do not result in an amino acid change in the encoded protein. Codon optomisation provides for improved expression of the nucleic acid in mammalian cells and/or conveys less immunogenicity. Immune optimisation involves substitution of one or more amino acids (i.e. see Table 1b), for example from one to ten amino acids, in the protein to provide a variant protein that exhibits reduced immunogenicity in-vivo in humans compared to yeast NDI1 protein. Examples of possible amino acid changes include conservative amino acid changes at one or more of the following positions:
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259, K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85, Y151, Y482, S488, V45, L483, S80, K196, for example one or more of the following amino acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
In a first aspect, the invention provides an isolated nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a functional variant thereof having at least 90% sequence identity with SEQ ID NO: 542, wherein the nucleic acid comprises at least 50 codons which are codon optimised compared with the sequence of yeast NDI1 gene of SEQ ID NO: 1.
Examples of codon optimised variants of yeast NDI1 gene are provided in SEQ ID NO'S: 2-62, 75-145, 165-243, 264-341, 362-441, 462-541, and 705-1004.
In a second aspect, the invention provides an isolated codon optimised nucleic acid sequence encoding an immune optimised functional variant of the yeast NDI1 protein of SEQ ID NO: 542 comprising at least one conservative amino acid change at a residue selected from the group consisting of:
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259, K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85, Y151, Y482, S488, V45, L483, S80, K196, for example one or more of the following amino acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T, wherein the nucleic acid comprises at least 50 codons which are codon optimised compared with the sequence of wild-type yeast NDI1 gene of SEQ ID NO: 1.
Examples of immune and codon optimised variants of yeast NDI1 gene are provided in SEQ ID NO'S: 75-145, 165-243, 264-341, 362-441, 462-541, 566-584, 705-824, 835-884, 895-944 and 955-1004.
In a third aspect, the invention provides an isolated nucleic acid sequence encoding an immune optimised functional variant of yeast NDI1 protein of SEQ ID NO: 542 in which the variant comprises at least one conservative amino acid change at a residue selected from the group consisting of:
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259, K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85, Y151, Y482, S488, V45, L483, S80, K196, for example one or more of the following amino acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T,
In an additional aspect of the invention the NDI1 gene and encoded protein are immune optimized employing amino acid substitution(s) at one or more key NDI1 positions as defined by K10, L19, V45, S80, I82, R85, S86, L89, F90, L94, S143, L150, Y151, L159, L195, K196, L202, K214, L259, V266, K284, K373, A387, L403, R479, L481, Y482, L483, S488, L502, K511.
Examples of immune optimised variants of yeast NDI1 gene (without codon optimisation) are provided in SEQ ID NO'S: 63-74 and 547-565 (one amino acid change), 146-164 and 585-605 (two amino acid changes), 244-263 and 606-640 (three amino acid changes), 641-675 (four amino acid changes), 342-361 and 676-696 (five amino acid changes), 697-703 (six amino acid changes), 704 (seven amino acid changes) and 442-461 (ten amino acid changes).
Typically, the nucleic acid sequence of the invention encodes a functional variant of the yeast NDI1 protein of SEQ ID NO: 542 having at last 90% sequence identity with SEQ ID NO:542. Preferably, the functional variant comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 542.
Preferably, the nucleic acid sequence of the invention encodes a yeast NDI1 protein that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically, from 1-20, 1-15, or ideally from 1-10, amino acids are changed. The changes are suitably conservative changes made to one or more of the residues identified above, for example one or more of: L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
Preferably, the nucleic acid sequence of the invention encodes a yeast NDI1 protein that includes at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically, from 1-20, 1-15, or ideally from 1-10, amino acids are changed, and the changes are suitably selected at NDI1 positions from the group: K10, L19, V45, S80, I82, R85, S86, L89, F90, L94, S143, L150, Y151, L159, L195, K196, L202, K214, L259, V266, K284, K373, A387, L403, R479, L481, Y482, L483, S488, L502, K511.
Suitably, the variant protein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of the amino acid changes selected from: L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
Ideally, the variant protein includes an amino acid change selected from: L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I, L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
Preferably, at least 90, 100, 150, 200, 250, 300, 320, or 329 codons are codon optimised for use in a mammal. In one embodiment, 1-100, 100-200, 200-300, or 300-329 codons are optimised. Ideally, 329 codons are optimised (see SEQ ID NO's 62, 134-145, 225-243, 324-341, 422-441, 522-541, 566-584 and 705-824).
In another embodiment 1-100, 100-200, 200-300, or 300-329 NDI1 codons are optimised for use in mammals and the nucleic acid sequence encodes a yeast NDI1 protein that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically, from 1-20, 1-15, or ideally from 1-10, amino acids are changed, and the changes are suitably selected at NDI1 positions from the group: K10, L19, V45, S80, I82, R85, S86, L89, F90, L94, S143, L150, Y151, L159, L195, K196, L202, K214, L259, V266, K284, K373, A387, L403, R479, L481, Y482, L483, S488, L502, K511.
Preferably, the nucleic acid of the invention encodes a variant protein having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 542.
The invention also relates to a nucleic acid construct comprising a nucleic acid sequence of the invention and a nucleic acid sequence encoding a mitochondrial localisation sequence. This may be, but are not limited to, sequences such as MLSKNLYSNKRLLTSTNTLVRFASTRS (SEQ ID NO: 1006) or MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 1007).
The invention also relates to a nucleic acid construct encoding a protein of the invention. The nucleic acid may be a DNA or RNA nucleic acid. The nucleic acid of the invention may use modified nucleic acids to optimise delivery and or increase stability and or increase longevity and or reduce immunogenicity 22,23.
In one aspect the invention relates to delivery of RNA encoding the protein and or protein variants of the invention.
The invention also relates to a protein encoded by a nucleic acid construct of the invention.
The term “nucleic acid sequence of the invention” as employed hereafter should be understood to mean either or both of the nucleic acid sequences of the invention and the nucleic acid constructs of the invention.
The invention also relates to a nucleic acid sequence selected from SEQ ID NO's: 1-541 and 547-1004.
The invention also relates to a protein encoded by a nucleic acid sequence of the invention. The protein may also include one or more mitochondrial localisation signal(s). This may be but not limited to sequences such as MLSKNLYSNKRLLTSTNTLVRFASTRS (SEQ ID NO: 1006) or MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 1007).
The invention also relates to a vector suitable for use in gene therapy and comprising a nucleic acid sequence of the invention. Suitably the vector is a viral vector, typically an adeno-associated virus (AAV), preferably AAV virus serotype 2, although other AAV serotypes and other types of vectors may be employed such as for example other viral vectors, non-viral vectors, naked DNA and other vectors, examples of which are listed in Table 5. Typically, the nucleic acid of the invention is expressed singly from the vector (single delivery vehicle). In another embodiment, the nucleic acid of the invention is expressed together with another gene either from the single delivery vehicle or using two delivery vehicles, for example, a gene that enhances cell survival and or cell function such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others, examples of which are described in Table 6. Genes may be delivered at the same time and/or before and/or after each other. Ideally, the second gene is a neurotrophic factor, examples of which are described in Table 6.
The invention also relates to a kit comprising a vector of the invention in combination with a second vector comprising a gene that enhances cell survival and or cell function such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others, examples of which are described in Table 6. Ideally, the second vector comprises a gene encoding a neurotrophic factor.
In an additional aspect additional gene sequences may be expressed in the same vector as the nucleic acid of the invention from a component such as an internal ribosome entry site (IRES) and or may be expressed using two or multiple promoter sequences.
Typically, the vector of the invention comprises a promotor wherein the nucleic acid of the invention is expressed from the promotor. Preferably, the promotor is one that is preferentially or specifically expressed in retinal ganglion cells (RGC's) wherein expression of the nucleic acid of the invention is under the control of the promotor. Examples of such promotors are described in Table 4. In an alternative embodiment, the vector of the invention comprises a promotor known to be expressed at low levels in RGC's.
In a further embodiment, the promotor is one that is known to be expressed in multiple cell types, examples of which are described in Table 4.
In an additional aspect, the nucleic acid of the invention is expressed from an inducible and/or conditional promotor.
In a further embodiment, the promotor is a tissue specific and/or cell specific promotor targeting mammalian cells other than RGC's such as the rhodopsin promotor which expresses in rod photoreceptor cells. Suitably, the vector comprises tissue specific and/or cell specific promotors combined with an inducible promotor system to control expression of the nucleic acid.
The promotors may control expression of the nucleic acid of the invention in combination with additional genes, as described above. Alternatively, the vector may comprise different promotors for expressing the nucleic acid of the invention and the other genes, for example, a gene encoding a neurotrophic agent.
The invention also relates to a method for the treatment and/or prevention of a neurodegenerative disease, especially LHON, which method comprises a step of delivering a nucleic acid of the invention to an individual by means of intraocular, ideally intravitreal, delivery. In one aspect a nucleic acid of the invention is delivered to an individual by means of systemic administration.
Preferably, the step of delivering the nucleic acid of the invention involves delivering a vector of the invention to the individual.
The invention also relates to the use of a nucleic acid of the invention, or a protein encoded by a nucleic acid of the invention, or a vector of the invention, as a medicament.
The invention also relates to a nucleic acid sequence of the invention, or a protein encoded by a nucleic acid sequence of the invention, or a vector of the invention, for use in the treatment of a disease or condition associated with mitochondrial dysfunction, for example a neurodegenerative disease, especially Leber Hereditory Optic Neuropathy (LHON). Typically, the treatment is symptomatic or prophylactic treatment.
The invention also relates to a method of treating a disease, for example a disease associated with mitochondrial dysfunction, for example a neurodegenerative disease, in an individual comprising a step of administering an active agent to the individual, typically administering the active agent to the eye, ideally to the retinal ganglion cells, photoreceptor cells or other eye cells, in which the active agent includes a nucleic acid sequence of the invention, a protein encoded by the nucleic acid sequence of the invention, or a vector of the invention. The treatment may be symptomatic or prophylactic treatment.
Typically, the active agent is administered by intra-ocular, ideally intra-vitreal and/or subretinal, administration. The active agent may include an additional agent, for example a gene or protein or compounds that enhances cell survival and or cell function such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others, examples of which are described in Table 6. The active agent and the additional agent, for example an additional gene, may be delivered at the same time or before or after each other.
Ideally, the additional agent is a gene encoding a neurotrophic factor, examples of which are described in Table 6. The active agent may be delivered by means of a vector, or by means of separate vectors, or by direct delivery of the additional agent. The active agent may be delivered to other parts of the body involving mitochondrial dysfunction, for example, to the brain for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease or dementia, or to photoreceptor cells for the treatment of Retinitis Pigmentosa or Age-related macular degeneration, or to muscle cells to treat muscle weakness and/or degeneration.
Further, the nucleic acid sequence of the invention, its protein product, or a vector of the invention, may be delivered to the target cell or tissue at the same time or at a different time to the additional agent.
The invention also relates to a cell, for example a stem cell or progenitor cell, RGC or RGC precursor cell that is transformed with a nucleic acid of the invention. Cells of the invention may be delivered to the eye via subretinal and/or intravitreal injection to treat cells of the eye affected by mitochondrial dysfunction such as RGC dysfunction. Alternatively, cells of the invention may be delivered to other parts of the body involving mitochondrial dysfunction, for example to the brain for the treatment of neurodegenerative diseases such as Alzheimer disease, Parkinsons disease or dementia, or to photoreceptor cells for the treatment of Retinitis Pigmentosa or Age-related macular degeneration, or to muscle cells to treat muscle weakness and/or degeneration.
Thus, the invention also relates to a transformed cell of the invention for use as a medicament. The invention also relates to a method of treating a disease or condition involving mitochondrial dysfunction, typically a neurodegenerative disease, suitably LHON, comprising a step of delivering cells of the invention to the individual.
The invention also provides a pharmaceutical formulation comprising an active agent selected from a nucleic acid of the invention, a protein encoded by the nucleic acid of the invention, a vector of the invention, or a cell of the invention, in combination with a pharmaceutically acceptable carrier.
Suitably, the formulation is provided in the form of a slow release capsule adapted to release the active agent following subretinal and or intravitreal injection, or following delivery to or close to a target tissue type/cell type (see examples in Table 7).
In an additional embodiment encapsulated cell technology is employed for delivery of the therapy.
In one embodiment the invention provides a transgenic organ, or a transgenic non-human animal, comprising the nucleic acids and vectors of the invention.
In another embodiment the invention may be delivered to cells with mutations in the nuclear genome which lead to disease phenotypes which are similar to disease phenotypes related to mitochondrial mutations. For example the disease phenotypes described in Table 8 may all result from nuclear mutations or mitochondrial mutations and hence may benefit from the invention. The invention would need to be delivered to the appropriate affected cell or tissue type. Typically these nuclear mutations affect cell types that require high levels of energy such as neurons and muscle cells. Hence these disorders, resulting from mutations in the nuclear genome and affecting these high energy requiring cell types may also benefit from additional energy provided by the invention.
In a further aspect, the invention relates to a method for the treatment or prevention of a neurodegenerative disease, especially LHON, which method comprises a step of delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention, to an individual by means of intraocular delivery, ideally intravitreal and/or subretinal delivery.
In a yet further aspect, the invention relates to a method for the treatment or prevention of a neurodegenerative disease, especially LHON, which method comprises a step of delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention, and an agent that enhances cell survival and or cell function such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others (examples of which are described in Table 6) to an individual. Treatment may be symptomatic or prophylactic.
In a yet further aspect, the invention relates to a method for the treatment or prevention of a neurodegenerative disease, especially LHON, which method comprises a step of delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention using an AAV vector, and delivery of an agent, using the same or a separate AAV vector, that enhances cell survival and or cell function such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others (examples of which are described in Table 6) to an individual. Treatment may be symptomatic or prophylactic.
The term “yeast NDI1 gene” refers to the wild-type Saccharomyces cerviscae NDI1 gene shown in SEQ ID NO: 1.
The term “variant of yeast NDI1 gene” means a variant of yeast NDI1 gene which differs from the wild-type gene due to at least codon optimisation, immune optimisation, or both.
The term “conservative amino acid change” should to be understood to mean that the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. In particular, it refers to the substitution of an amino acid of a particular grouping as defined by its side chain with a different amino acid from the same grouping.
The term nucleic acid means deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and artificial nucleic acid analogs such as peptide nucleic acid (PNA), morpholino- and locked nucleic acid, glycol nucleic acid and threose nucleic acid. Artificial nucleic acid analogs differ from DNA and RNA as they typically contain changes to the backbone of the molecule. Nucleic acids incorporating chemical modification(s) to DNA and RNA to optimise delivery and or increase stability and or increase longevity and or reduce immunogenicity are also contemplated by the term nucleic acid. Modifications, such as phosphorothioates, boranophosphate, 2′-Amino, 2′-Fluoro, 2′-Methoxy have been made to nucleic acids to modulate parameters such as resistance to nuclease degradation, binding affinity and or uptake. Exemplary nucleic acid molecules for use are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA and or RNA. Modifications include but are not limited to inclusion of 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 2′-O-methyl, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), -5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine, 2-thiourdine, 5-methyl-cytidine amongst others.
The term “codon optimised” means that a codon that expresses a bias for yeast (i.e. is common in yeast genes but uncommon in mammalian genes) is changed to a synonomous codon (a codon that codes for the same amino acid) that expresses a bias for mammals. Thus, the change in codon does not result in any amino acid change in the encoded protein.
The term “immune optimised” as applied to a variant of yeast NDI1 gene means that the gene variant encodes a variant NDI1 protein which elicits a reduced immune response when expressed in a mammal compared to the wild-type yeast NDI1 gene.
The term “yeast NDI1 protein” should be understood to mean the wild-type Saccharomyces cerviscae NDI1 protein shown in SEQ ID NO: 542. The “functional variant” should be understood to mean a variant of SEQ ID NO: 542 which retains the functionality of yeast NDI1 protein, for example, comparable oxygen consumption measurements in the presence of rotenone (see methods below/
The term “neurodegenerative disease” should be understood to mean a disease characterised by neuronal injury or death, or axonal degeneration, and includes diseases such as motor neuron disease; prion disease; Huntington's disease; Parkinson's disease; Parkinson's plus; Tauopathies; Chromosome 17 dementias; Alzheimer's disease; Multiple sclerosis (MS); hereditary and acquired neuropathies; retinopathies and diseases involving cerebellar degeneration.
In the context of the present invention, the term “gene therapy” refers to treatment of individual which involves insertion of a gene into an individual's cells for the purpose of preventing or treating disease. Insertion of the gene is generally achieved using a delivery vehicle, also known as a vector. Viral and non-viral vectors may be employed to deliver a gene to a patients' cells. Other types of vectors suitable for use in gene therapy are described below.
The term “neurotrophic agent” should be understood to mean a protein that induces the survival, development and function of neurons. Examples include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Other examples are provided below.
Retinal ganglion cells (RGCs) are types of neurons located close to the inner surface (the retinal ganglion layer) of the retina of the eye. They collectively image forming and non-image forming visual information from the retina to several regions in the thalamus, hypothalamus, and mid-brain.
It will be appreciated that the nucleci acids of the invention may include one or more polyadenylation signals, typically located at the 3′-end of the molecule. In addition, the nucleic acid may include a leader sequence and/or a stop codon. It will also be appreciated that the nucleci acids of the invention may include one or more signals to facilitate import of proteins into mitochondria.
Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).
Apart from the specific delivery systems embodied below, various delivery systems are known and can be used to administer the therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In addition, naked DNA can be used for delivery.
In one aspect of the invention, agents such as surfactants may be included in formulations to minimize aggregation of the therapeutic of the invention, whether viral and/or non-viral vectors, proteins or polypeptides and/or cells.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved, for example, by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed., Eng. 14:201 (1987); Buchwald et al., Surgery 88:75 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
The present invention also provides pharmaceutical compositions comprising a nucleic acid of the invention and/or a protein encoded by the nucleic acid. Such compositions comprise a therapeutically effective amount of the therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to, ease pain at the, site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circ*mstances.
Nucleic Acid Sequences of the Invention
The sequence listing below provides a number of nucleic acid sequences according to the invention, specifically:
SEQ ID NO: 1—Yeast NDI1 gene—0 amino acid changes—0 codon changes
SEQ ID NO'S 2-21 and 825-834—Yeast NDI1 gene—0 amino acid changes—100 codon changes
SEQ ID NO'S 22-41 and 885-894—Yeast NDI1 gene—0 amino acid changes—200 codon changes
SEQ ID NO'S 42-61 and 945-954—Yeast NDI1 gene—0 amino acid changes—300 codon changes
SEQ ID NO 62—Yeast NDI1 gene—0 amino acid changes—329 codon changes
SEQ ID NO'S 63-74 and 547-565—Yeast NDI1 gene—1 amino acid changes—0 codon changes
SEQ ID NO'S 75-94 and 835-844—Yeast NDI1 gene—1 amino acid changes—100 codon changes
SEQ ID NO'S 95-114 and 895-904—Yeast NDI1 gene—1 amino acid changes—200 codon changes
SEQ ID NO'S 115-134 and 955-964—Yeast NDI1 gene—1 amino acid changes—300 codon changes
SEQ ID NO'S 134-145 and 566-584—Yeast NDI1 gene—1 amino acid changes—329 codon changes
SEQ ID NO'S 146-164 and 585-605—Yeast NDI1 gene—2 amino acid changes—0 codon changes
SEQ ID NO'S 165-184 and 845-854—Yeast NDI1 gene—2 amino acid changes—100 codon changes
SEQ ID NO'S 185-204 and 905-914—Yeast NDI1 gene—2 amino acid changes—200 codon changes
SEQ ID NO'S 205-224 and 965-974—Yeast NDI1 gene—2 amino acid changes—300 codon changes
SEQ ID NO'S 225-243 and 705-725—Yeast NDI1 gene—2 amino acid changes—329 codon changes
SEQ ID NO'S 244-263 and 606-640—Yeast NDI1 gene—3 amino acid changes—0 codon changes
SEQ ID NO'S 264-283 and 855-864—Yeast NDI1 gene—3 amino acid changes—100 codon changes
SEQ ID NO'S 284-303 and 915-924—Yeast NDI1 gene—3 amino acid changes—200 codon changes
SEQ ID NO'S 304-323 and 975-984—Yeast NDI1 gene—3 amino acid changes—300 codon changes
SEQ ID NO'S 324-341 and 726-760—Yeast NDI1 gene—3 amino acid changes—329 codon changes
SEQ ID NO'S 641-675—Yeast NDI1 gene—4 amino acid changes—0 codon changes
SEQ ID NO'S 865-874—Yeast NDI1 gene—4 amino acid changes—100 codon changes
SEQ ID NO'S 925-934—Yeast NDI1 gene—4 amino acid changes—200 codon changes
SEQ ID NO'S 985-994—Yeast NDI1 gene—4 amino acid changes—300 codon changes
SEQ ID NO'S 761-795—Yeast NDI1 gene—4 amino acid changes—329 codon changes
SEQ ID NO'S 342-361 and 676-696—Yeast NDI1 gene—5 amino acid changes—0 codon changes
SEQ ID NO'S 362-381 and 875-884—Yeast NDI1 gene—5 amino acid changes—100 codon changes
SEQ ID NO'S 382-401 and 935-944—Yeast NDI1 gene—5 amino acid changes—200 codon changes
SEQ ID NO'S 402-421 and 995-1004—Yeast NDI1 gene—5 amino acid changes—300 codon changes
SEQ ID NO'S 422-441 and 796-816—Yeast NDI1 gene—5 amino acid changes—329 codon changes
SEQ ID NO'S 697-703—Yeast NDI1 gene—6 amino acid changes—0 codon changes
SEQ ID NO'S 817-823—Yeast NDI1 gene—6 amino acid changes—329 codon changes
SEQ ID NO 704—Yeast NDI1 gene—7 amino acid changes—0 codon changes SEQ ID NO 824—Yeast NDI1 gene—7 amino acid changes—329 codon changes
SEQ ID NO'S 442-461—Yeast NDI1 gene—10 amino acid changes—0 codon changes
SEQ ID NO'S 462-481—Yeast NDI1 gene—10 amino acid changes—100 codon changes
SEQ ID NO'S 482-501—Yeast NDI1 gene—10 amino acid changes—200 codon changes
SEQ ID NO'S 502-521—Yeast NDI1 gene—10 amino acid changes—300 codon changes
SEQ ID NO'S 522-541—Yeast NDI1 gene—10 amino acid changes—329 codon changes
SEQ ID NO: 542—Yeast NDI1 protein—0 amino acid changes
Notably OphNDI1 may contain 0-10 amino acid substitutions to modulate and immune response or 1-329 altered codons, which are expressed more frequently in mammalian cells than the wild type codons in NDI1 (Table 1a & 1b and Sequence Listing). In addition the CMV and ubiquitin promoters may be substituted for any of the promoters indicated in Tables 2-4 and the GDNF gene may be substituted for any gene indicated in Table 6. Sequences for these core construct designs are presented in Table 1a & 1b and the attached Sequence Listing. Notably, different polyadenalation signals may also be utilised in the constructs described.
A, Levels of NDI1 expressed from unmodified vector (NDI1) and from NSG, which expresses both an unmodified NDI1 gene and a GDNF gene, were compared by real time RT-PCR. Levels of expression (y-axis) are expressed in copy number per unit of the housekeeping gene, β-actin.
B, Levels of humanised NDI1 (hNDI1) expressed in mouse retina delivered invitreally using AAV2/2 vectors were compared to levels of unmodified NDI1 delivered also using AAV2/2. Levels of expression are expressed in copy number per unit of the housekeeping gene β-actin. As expression levels in
C, RT-PCR samples performed on RNA samples extracted from wild type mice which were intravitreally injected with AAV2/2 vectors expressing variants of the NDI1 gene and run on 3% agarose gels. Lanes 1 and 8, GeneRuler 100 bp DNA size ladder (Fermentas). The two lower bands of the ladder represent 100 and 200 bp. Lane 2, NDI1; Lane 3, NSG; Lane 4, NDI1 with V266I modification; Lane 5, NSG; Lane 6, humanised NDI1; Lane 7 Humanised NDI1 with I82V modification. NDI1 amplification product is 87 bp and humanised NDI1 amplification product is 115 bp. Equal amounts of PCR products were loaded into each well. The hNDI1 and NSG vectors resulted in visibly higher levels of expression than the unmodified NDI1 vector mirroring the findings in
In the present invention delivery of NDI1 constructs (
In the present Application, intravitreal injection of AAV-NDI1 provided substantial protection against rotenone-induced insult, as assessed by a variety of assays (
Following the successful delivery of AAV-NDI1 to RGCs using intravitreal injection, NDI1 was codon optimised so that codons which are used more frequently in mammalian cells were introduced to the NDI1 yeast gene. Codon modifications from 1-329 codons can be implemented to optimize expression of NDI1 in mammals while maintaining wild type amino acids. The maximal number of codons that can be altered in NDI1 to align codons with those most frequently used in mammals is 329 codons and these alterations were employed to generate a construct termed OphNDI1 and also known as humanized NDI1 (hNDI1). Plasmids containing OphNDI1 (hNDI1) or wild type NDI1, both expressed from a cytomegalovirus (CMV) promoter and containing a minimal polyadenylation (PolyA) signal, a modified rabbit beta-globin polyadenylation signal, were transiently transfected into HeLa cells using lipofectamine. Levels of NDI1 protein expression from NDI1 and hNDI1 constructs were compared using Western Blot analysis. hNDI1 (OphNDI1) was determined to express more highly than wild type NDI1 indicating that codon optimising the NDI1 gene has indeed enhanced expression in mammalian cells (
In addition both the wild type and the codon-optimised NDI1 constructs have been immuno-optimised by introducing one or more amino acid changes to modulate the immune response(s) (Table 1a & 1b and Sequence Listing). Amino acid modifications were undertaken subsequent to in silico analyses for potential immunogenic sites within NDI1 (see
In addition, to codon-optimized and immuno-optimized NDI1 constructs, a dual component construct was generated containing the CMV promoter driven NDI1 gene together with a ubiquitin promoter driven glial derived neurotrophic factor (GDNF) gene (NSG), the latter employing a neurturin polyA signal (
Cohorts of adult wild type mice were intravitreally injected with 3 ul of AAV2/2 vectors expressing either NDI1, hNDI1, immuno-optimised hNDI1 I82V, immuno-optimised NDI1 V266I or AAV-NSG. Two weeks post-injection retinas were harvested from treated mouse eyes and total RNA extracted. Levels of expression from AAV vectors in mouse retinas were evaluated by real time RT-PCR (
All gene therapies which deliver non-human proteins risk activation of cytotoxic T-cell responses following presentation of peptide fragments derived from the transgenic protein. It is therefore important to the success of the treatment that immunogenicity of the transgenic protein is modulated. One of the most effective ways this can be done is by searching the sequence of the protein for fragments which are likely to strongly bind MHC-I, increasing the likelihood that they will be presented on the cell surface and so induce an immune reaction.
This approach is complicated somewhat by the presence of many different MHC-I alleles in the human population, each of which may have slightly different binding affinities for different peptides.
There are established bioinformatics methods for predicting the MHC-I binding affinity of a particular peptide, several of which are available as downloadable tools. For our purposes, the consensus prediction method of Nielsen et al (Protein Sci. 2003 May; 12(5):1007-17) was most suitable, in addition to having excellent experimentally-validated accuracy. These tools were adapted and supporting software generated to enable prediction of affinity for a wide variety of MHC-I alleles. The computational tool thus generated may be applied and modified to predict other types of immune responses.
All potential peptide fragments that could be derived from the Ndi1 protein were assayed by the consensus prediction method for binding affinity to all well-characterised human MHC-I proteins.
Methods
Vector Construction and AAV Production
Yeast NDI1 (Accession No: NM_001182483.1) was cloned as described53. Briefly, NDI1 was PCR amplified from total yeast DNA extracted from S288c using the following primers F: TTCTCGAGGTAGGGTGTCAGTTTC (SEQ ID NO: 543) and R: AAAGCGGCCGCAGTGATCAACCAATCTTG (SEQ ID NO: 544) and cloned into XhoI and NotI sites of pcDNA3.1- (Invitrogen, Paisley, UK). A minimal poly-adenylation signals4 was cloned downstream of NDI1 using NotI and EcoRV. The CMV immediate early promoter (present in pcDNA3.1-), the NDI1 gene and poly-adenylation signal were isolated on a MluI and EcoRV fragment, end filled and cloned into the NotI sites of pAAV-MCS (Agilent Technologies, La Jolla, Calif., USA) to create pAAV-NDI;
The entire human GDNF coding sequence from the atg start codon (nucleotides 201-836 of accession number NM_000514) was cloned 3-prime of a 347 bp human Ubiquitin promoter (nucleotides 3557-3904 of accession number D63791) and a human Neurturin polyA consisting of nucleotides 1057-1160 of accession number AL161995 was cloned down-stream of the GDNF gene. This entire ubiquitin-driven GDNF cassette, including Neurturin polyA was cloned downstream of the CMV-driven NDI1 (including the rabbit b-globulin polyA).
Codon optimized NDI1 sequences and/or with amino acid changes to reduce immunogenicity profiles were synthesized by Geneart Inc. These were isolated on a XbaI and XhoI fragment and cloned into pAAV-MCS (Agilent Technologies, La Jolla, Calif., USA) and pcDNA3.1- (Invitrogen, Paisley, UK) plasmids with a CMV immediate early promoter and minimal polyA and verified by DNA sequencing.
Recombinant AAV2/2 viruses, AAV-ND1, AAV-NSG, pAAV-NDI1 V266I, AAV-huNDI1, pAAV-huNDI1 182V and AAV-EGFP were prepared as described20, with a modified cesium chloride gradient as described19 Additional AAV-ND1, AAV-NSG recombinant AAV2/2 viruses were generated by the Gene Vector production Center of Nantes. Genomic titres (DNase-resistant viral particles per milliliter; vp/ml) were determined by quantitative real-time-polymerase chain reaction (qRT-PCR) according to the method of Rohr et al.21
Cell Culture
Human cervical carcinoma cells (HeLa, ATCC accession no. CCL-2) were transfected with pAAV-NDI1 or pAAV-EGFP using Lipofectamine 2000 reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., USA). 5×105 cells per well were seeded onto 6-well plates containing 1 ml Dulbecco's modified Eagle medium supplemented with 10% calf serum, 2 mM glutamine and 1 mM sodium pyruvate and incubated overnight at 37° C. Media was then aspirated and the cells were washed twice with phosphate-buffered saline (PBS). Each well was transfected with 1 μg pAAV-NDI1 or 1 μg pAAV-EGFP in triplicate. Cells were harvested 48 hrs later and the cells from each triplicate pooled for an individual experiment, each experiment was repeated in triplicate.
Mitochondrial Isolation and Western Blot Analysis
Mitochondria were isolated from HeLa cells using Anti-TOM22 microbeads (Mitochondria isolation kit, Miltenyi Biotec GmbH, Germany). Isolated mitochondria were washed twice in PBS and hom*ogenised in 100 μl radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TrisCl pH 8.0 and 1 protease inhibitor co*cktail tablet/10 mls (Roche, Mannheim, Germany)). The hom*ogenate was centrifuged at 10,000 g for 20 min at 4° C. and the supernatant removed for analysis. Normalised protein samples were separated on 12% polyacrylamide gels and electrophoretically transferred to PVDF membranes (Bio-Rad, Berkley, Calif., USA). The PVDF membrane was blocked with 5% non-fat milk in tris buffered saline (TBS, 0.05M Tris, 150 mM NaCl, pH 7.5) and 0.05% (vol/vol) Tween 20 for 1 hr at room temperature. Rabbit polyclonal antibodies to NDI1 (1:500, Cambridge Research Biochemicals, Cleveland, UK) and VDAC1 (1:1000, Abcam, Cambridge, UK) were diluted in 5% milk and incubated overnight at 4° C. Membranes were washed twice with TBS and incubated with a secondary anti-rabbit (IgG) horseradish peroxidise-conjugated antibody (1:2500, Sigma-Aldrich, St. Louis Mo., USA) for 2 hr at room temperature, exposed to Super-Signal chemiluminescent substrate and enhancer (Pierce Biotechnology, Rochford, Ill., USA) and signal detected using X-ray film (Kodak, Rochester, N.Y., USA). All Western blots were repeated three times.
Respiratory Analysis
Respiratory measurements were performed in DMEM at 37° C. on an Oxygraph-2k (OROBOROS® INSTRUMENTS GmbH, Innsbruck, Austria) according to the manufacturer's instructions. Briefly, each chamber was calibrated with 2 mls DMEM and stirred (200 rpm) for 1 hr to saturate the media with oxygen. Parallel experiments were run in the two chambers of the Oxygraph-2k using 1×106 pAAV-NDI1 or 1×106 pAAV-EGFP transfected HeLa cells. Following the addition of cells to the oxygen saturated media the chamber size was reduced to 2 ml to remove air. Continuous readings were taken to establish the fully oxygenated baseline. 2 ul 5 mM rotenone (5 μM in 100% ethanol) was added to 1×106 pAAV-NDI1 or 1×106 pAAV-EGFP transfected HeLa cells prior to transfer to the requisite chambers and continuous post-rotenone readings taken. Continuous readings were taken both with and without rotenone until oxygen consumption stabilised. Readings were taken from three independent transfections for each construct.
Animals and Intravitreal Injections
Wild type 129 S2/SvHsd (Harlan UK Ltd, Oxfordshire, UK) mice were maintained under specific pathogen free (spf) housing conditions. Intravitreal injections were carried out in strict compliance with the European Communities Regulations 2002 and 2005 (Cruelty to Animals Act) and the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals. Briefly, adult mice were anaesthetised and pupils dilated as described57. Using topical anaesthesia (Amethocaine), a small puncture was made in the sclera. A 34-gauge blunt-ended microneedle attached to a 10 μl Hamilton syringe was inserted through the puncture, and 0.6 μl 2.5 mM rotenone (1.5 nmol) in dimethyl sulfoxide (DMSO, vehicle), 0.6 μl DMSO alone or 3 μl 1×1012 vp/ml AAV2/2 was slowly, over a two minute period, administered into the vitreous. Following intravitreal injection, an anesthetic reversing agent (100 mg/10 g body weight; Atipamezole Hydrochloride) was delivered by intraperitoneal injection. Body temperature was maintained using a homeothermic heating device. All animal studies have been approved by the authors' Institutional Review Board.
RNA Extraction and PCR Analysis
Adult wild type mice (n=6) were intravitrally injected with 3×109 vp AAV-NID1 while fellow eyes received 3×109 vp AAV-EGFP. Retinas were harvested two weeks post-injection and total RNA extracted using the QIAGEN RNEASY™ Qiagen RNeasy kit according to the manufacturer's specification. In vivo expression of NDI1 from AAV-NDI1 was confirmed by reverse transcription PCR (RT-PCR) on a 7300 Real Time PCR System (APPLIED BIOSYSTEMS™, Foster City, Calif., USA) and resulting amplification products separated and sized on 2.5% agarose gels. The following primers were used: NDI1 forward primer 5′ CACCAGTTGGGACAGTAGAC 3′ (SEQ ID NO: 545) and NDI1 reverse primer: 5′ CCTCATAGTAGGTAACGTTC 3′ (SEQ ID NO: 546). Humanised forms of NDI1 transcript were RT-PCR amplified with hNDI1 forward primer 5′ GAACACCGTGACCATCAAGA 3′ (SEQ ID NO: 1008) and hNDI1 reverse primer 5′ GCTGATCAGGTAGTCGTACT 3′(SEQ ID NO: 1009). β-actin was used as an internal control as described (ref). RT-PCRs were performed twice in triplicate or quadruplicate. Levels of NDI1 or humanized NDI1 expression were determined by real time RT PCR using the QUANTITECT™ SYBR green RT PCR kit (QIAGEN™). Briefly, the copy number of two plasmid DNA preparations containing either NDI1 or humanized NDI1 was determined by spectraphotometry on a NanoDrop and serial dilutions of these plasmid DNA preparations were prepared containing between 10e2-10e7 copies/μl. These standard curves were included in 96-well plates that also included RNA samples to be analysed. Hence expression levels from all constructs, whether humanized or not, could be compared using absolute copy number, even though the primer pairs used for non: humanized and humanized PCR amplification were not the same. Expression levels were normalized using the internal housekeeping gene β-actin.
Histology
Eyes and optic nerves were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at room 4° C. washed three times with PBS and cryoprotected using a sucrose gradient (10%, 20%, 30%). 10 μm sections were cut on a cryostat (HM 500 Microm, Leica, Solms, Germany) at −20° C. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Specimens were analysed with a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Corresponding microscope images taken with different filters were overlaid using Photoshop v. 10 (Adobe Systems Europe, Glasgow, UK). For ganglion cell (GCL) counts the ganglion cells were labelled using NeuN (Abcam, Cambridge, UK) immunohistochemistry as previously described. The primary antibody was diluted 1:100 and visualised using cy3-conjugated anti-mouse-IgG secondary antibody (Jackson ImmunoResearch Europe, Suffolk, UK). Four retinal sections per eye from four mice per group were analysed (n=4). The sections were taken approximately 150 μm apart in the central retina (600 μm span in total); 2 counts per section i.e. 8 counts per eye in total, were made using the count tool in Photoshop (Adobe systems). The diameter of the optic nerves was determined at approximately 5 mm from the optic nerve head from 3 animals per group (n=3). Three measurements per nerve were made approximately 150 μm apart using the ruler tool in Photoshop (Adobe Systems). Procedures for TEM were as previously described. Briefly, three weeks post-rotenone injection optic nerves were fixed in 4% paraformaldehyde in phosphate-buffered solution and fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.3) for 2 hr at room temperature. Washed specimens were post-fixed in buffered 2% osmium tetroxide, dehydrated and embedded in araldite. Ultrathin cross-sections were cut on a vibratome (Leica VT 1000 S), analysed using a Tecnai 12 BioTwin transmission electron microscope (FEI, Eindhoven, Holland) and imaged with a SIS MegaView III surface channel charge-coupled device (SCCD) camera (Olympus Soft Imaging Solutions, Münster, Germany). The total number of membrane debris particles in the images was counted in 5 cross sections per optic nerve from 3 animals per group (n=3).
Magnetic Resonance Imaging
Optic nerve integrity in experimental and control mice was assessed by Manganese (Mn2+) enhanced magnetic resonance imaging (MEMRI) technique using a 7 T Bruker Biospec 70/30 magnet (Bruker Biospin, Etlingen, Germany). MEMRI demarcates active regions of the brain due to the ability of Mn2+ ions to enter excitable cells through voltage-gated calcium channels, thus analysis of Mn2+ transport through the optic nerve provides a good measure of its integrity. Two hours prior to scanning, mice were anaesthetised and intravitreally injected, as described above, with 2 μl of 20 mg/ml manganese chloride solution. For image acquisition, mice were maintained under sedation with ketamine (375 μg/10 g body weight) and placed on an MRI-compatible cradle which maintains the animal's body temperature at 37° C. (respiration and temperature were monitored for the duration of experiment). The cradle was positioned within the MRI scanner and an initial rapid pilot image acquired to ensure accurate positioning of the mouse. Oblique coronal T1-weighted 2D images were acquired using FLASH sequence (TR/TE:150/2.5 ms; Matrix: 128×128; Field of View: 20×20 mm2; Flip Angle 50°; number of averages: 40, the pixel resolution was 0.156 mm/pixel). In the oblique coronal orientation (36°), 20 slices, each measuring 0.35 mm in thickness with 0.45 mm inter slice gap, were recorded for an acquisition time of 9 min 36 sec. MRI scans corresponding to the area immediately superior to the optic chiasm provided more consistent images compared to the optic nerve itself due to the variations in physically positioning each animal. Log signal intensities in this region were quantified using Image J© software (available on the world wide web at imagej.nih.gov/ij.
Optokinetics
Optokinetic response (OKR) spatial frequency thresholds were measured blind by two independent researchers using a virtual optokinetic system (VOS, OptoMotry, CerebralMechanics, Lethbridge, AB, Canada). OptoMotry36 measures the threshold of the mouse's optokinetic tracking response to moving gratings. Briefly, a virtual-reality chamber is created with four 17 inch computer monitors facing into a square and the unrestrained mouse was placed on a platform in the centre. A video camera, situated above the animal, provided real-time video feedback. The experimenter centred the virtual drum on the mouse's head and judged whether the mouse made slow tracking movements with its head and neck. The spatial frequency threshold, the point at which the mouse no longer tracked, was obtained by incrementally increasing the spatial frequency of the grating at 100% contrast. A staircase procedure was used in which the step size was halved after each reversal, and terminated when the step size became smaller than the hardware resolution (˜0.003 c/d, 0.2% contrast). One staircase was presented for each direction of rotation to measure each eye separately, with the two staircases being interspersed.
Statistical Analysis
Data sets of treated and untreated samples were pooled, averaged and standard deviation (SD) values calculated. Statistical significance of differences between data sets was determined by either Student's two-tailed t-test or ANOVA used with Tukey's multiple comparison post hoc test. In addition, the Kruskall-Wallis one-way analysis of variance was applied to the MRI data set and Mann Whitney U-tests were undertaken on all other data sets to establish that statistical significance was maintained using nonparametric statistical models. Analysis was performed using Prism v. 5.0 c (GraphPad Software, La Jolla, Calif., USA); differences with p<0.05 were considered statistically significant
Predictions of Immunogenic Codons
All potential peptide fragments that could be derived from the Ndi1 protein were assayed by the consensus prediction method for binding affinity to all well-characterised human MHC-I proteins. All epitopes displaying a high affinity for MHC-I (defined as a predicted IC50<500 nM) were noted, along with the corresponding MHC-I allele to which they had displayed high binding affinity. Each potential peptide fragment was then assigned an ‘immunogenicity score’, defined as the sum of the frequencies of all MHC-I alleles in the global human population for which it had a high binding affinity. The highest-scoring fragments were then selected for potential modification to reduce immunogenicity. All possible single amino acid mutations for each of these immunogenic fragment sequences were generated, and each was assayed for immunogenicity by the above methods. In addition, the BLOSUM62 matrix was used to calculate the sequence similarity between the original and mutated sequences. For each fragment, an optimal immunogenicity-reducing mutation was chosen. This was done by taking the set of all potential mutations for that fragment and eliminating all fragments which had an immunogenicity score greater than half of the immunogenicity score of the original fragment. The sequence with the highest sequence similarity to the original fragment (as defined by the BLOSUM62 matrix) was selected as the optimal substitution for that position.
In addition to the analyses described above using information regarding MHC-1 alone, immunogenicity estimation and reduction in Ndi1 was achieved via in silico modelling of antigen presentation via the MHC-I pathway using the IEDB proteasomal cleavage/TAP transport/MHC class I combined predictor.
As fragments of 9 amino acids in length are the most commonly presented fragments by MHC-I, all possible sequences of 9 consecutive amino acids that could be derived from Ndi1 were listed and passed to the IEDB predictor for analysis. For every 9-mer peptide P and MHC-I allele i, an immunogenicity value Gp,i was generated which is proportional to the amount of that fragment that would be displayed on the cell surface by a given MHC-I allele, taking into account proteasomal degradation, transport and binding by MHC-I.
An overall immunogenicity factor Fp for the 9-mer peptide was then calculated as
where Ni is the estimated prevalence of each allele in the global human population as a fraction of the total pool of alleles, calculated using population frequency data from The Allele Frequency Net Database (Gonzalez-Galarza et al, 2011). In other words, Fp represents the mean amount of that fragment that would be displayed on the surface of a cell for all MHC-I alleles, weighted by how frequently each allele occurs in the human population.
Each amino acid position A in the Ndi1 peptide was then assigned an immunogenicity score SA defined as the sum of the immunogenicity factors for all 9-mer peptides containing that amino acid. All positions whose immunogenicity score was less than one-fifth of the highest score were not considered further, as mutations at these positions would not be able to significantly affect the overall immunogenicity of the protein.
For each of the remaining positions, a BLOSUM matrix (Henikoff and Henikoff, 1992) was used to identify potential mutations that would not be overly disruptive to the structure or function of Ndi1. A BLOSUM matrix is calculated by aligning hom*ologous protein sequences from many species against each other, and comparing the frequency with which each amino acid is replaced by every other amino acid.
For two amino acids x and y, the BLOSUM score Bx,y is defined as the log-likelihood of the amino acid x replacing y or vice-versa in a given position in hom*ologous peptides. As a direct consequence of this definition, Bx,y=By,x for all x and y (in other words, all BLOSUM matrices are symmetric).
A high BLOSUM score for an amino-acid pair indicates that mutations changing one of those amino acids to the other are more likely to be observed in hom*ologous proteins, indicating that such changes are less likely to severely disrupt protein structure. A BLOSUM score can also be calculated between each amino acid and itself (Bx,x), indicating the likelihood that that amino acid will remain constant between hom*ologous proteins.
For all possible mutations at a given position, ΔB was defined as the change in the BLOSUM score for that mutation. More formally, given an initial amino acid x and a candidate replacement amino acid y, ΔB=Bx,x−Bx,y. All mutations for which ΔB was greater than 4 were considered too disruptive to protein function and not analysed further.
For all remaining candidate mutations, immunogenicity factors F and scores S were recalculated for the post-mutation peptide using the IEDB predictor. The reduction in immunogenicity ΔS was then determined, defined as the difference between the score S for that position in the original peptide versus the new score S after mutation.
All possible mutations were then ranked by the metric
High values of
represent mutations which are likely to cause a large reduction in immunogenicity with a relatively small predicted impact on protein function. Outputs with predicted amino acids and scores are provided in Table X.
The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
- 1. Yu-Wai-Man P, Griffiths P G, Chinnery P F. Mitrochondrial optic neuropathies—disease mechanisms and therapeutic strategies. Prog Retin Eye Res. 30:81-114 (2011).
- 2. Chalmers R M and Schapira A H. Clinical, biochemical and molecular genetic features of Leber's hereditary optic neuropathy. Biochim Biophys Acta. 1410 (2):147-158 (1999).
- 3. Mackey D A, Oostra R J, Rosenberg T, Nikoskelainen E, Bronte-Stewart J, Poulton J, Harding A E, Govan G, Bolhuis P A, Norby S. Primary pathogenic mtDNA mutations in multigeneration pedigrees with Leber hereditary optic neuropathy. Am J Hum Genet. 59 (2):481-485 (1996).
- 4. Jarrett S G, Lin H, Godley B F, Boulton M E. Mitrochondrial DNA damage and its potential role in retinal degeneration. Prog Retin Eye Res. 27 (6):596-607 (2008).
- 5. Ames A 3rd. CNS energy metabolism as related to function. Brain Res Brain Res Rev. 34:42-68 (2000).
- 6. Yen M Y, Wang A G, Wei Y H. Leber's hereditary optic neuropathy: a multifactorial disease. Prog Retin Eye Res. 25 (4):381-396 (2006).
- 7. Porter R K, Joyce O J, Farmer M K, Heneghan R, Tipton K F, Andrews J F, McBennett S M, Lund M D, Jensen C H, Melia H P. Indirect measurement of mitochondrial proton leak and its application. Int J Obes Relat Metab Disord. 23 (Suppl 6):512-18 (1999).
- 8. Yamada K, Mashima Y, Kigasawa K, Miyash*ta K, Wakakura M, Oguchi Y. High incidence of visual recovery among four Japanese patients with Leber's hereditary optic neuropathy with the 14484 mutation. J. Neuroophthalmol. 17 (2):103-107 (1997).
- 9. Marcuello A, Martinez-Redondo D, Dahmani Y, Casajús J A, Ruiz-Pesini E, Montoya J, López-Pérez M J, Díez-Sánchez C. Human mitochondrial varienats influence on oxygen consumption. Mitochondrion. 9:27-30 (2009).
- 10. Hudson G, Carelli V, Spruijt L, Gerards M, Mowbray C, Achilli A, Pyle A, Elson J, Howell N, La Morgia C, Valentino M L, Huoponen K, Savontaus M L, Nikoskelainen E, Sadun A A, Salomao S R, Belfort R Jr, Griffiths P, Man P Y, de Coo R F, Horvath R, Zeviani M, Smeets H J, Torroni A, Chinnery P F. Clinical expression of Leber hereditary optic neuropathy is affected by the mitrochondrial DNA-haplogroup background. Am J Hum Genet. 81:228-233 (2007).
- 11. Shankar S P, Fingert J H, Carelli V, Valentino M L, King T M, Daiger S P, Salomao S R, Berezovsky A, Belfort R Jr, Braun T A, Sheffield V C, Sadun A A, Stone E M. Evidence for a novel x-linked modifier locus for leber hereditary optic neuropathy. Ophthalmic Genet. 29 (1):17-24 (2008).
- 12. Tsao K, Aitken P A, Johns D R. Smoking as an aetiological factor in a pedigree with Leber hereditary optic neuropathy. Br J. Ophthalmol. 83 (5):577-581 (1999).
- 13. Giordano C, Montopoli M, Perli E, Orlandi M, Fantin M, Ross-Cisneros F N, Caparrotta L, Martinuzzi A, Ragazzi E, Ghelli A, Sadun A A, d'Amati G, Carelli V. Oestrogens ameliorate mitochondrial dysfunction in Leber's hereditary optic neuropathy. Brain. 134 (Pt 1):220-234 (2011).
- 14. Qi X, Sun L, Lewin A S, Hauswirth W W, Guy J. The mutant human ND4 subunit of complex I induces optic neuropathy in the mouse. Invest Ophthalmol Vis Sci. 48 (1):1-10 (2007).
- 15. Ellouze S, Augustin S, Bouaita A, Bonnet C, Simonutti M, Forster V, Picaud S, Sahel J A, Corral-Debrinski M. Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction. Am J Hum Genet. 83 (3):373-387 (2008).
- 16. Koilkonda R D, Guy J. Leber's Hereditary Optic Neuropathy-Gene Therapy: From Benchtop to Bedside. J Ophthalmol. 2011: 179412 [Epub ahead of print] (2011).
- 17. Marella M, Seo B B, Yagi T, Matsuno-Yagi A. Parkinson's disease and mitochondrial complex I: a perspective on the Ndi1 therapy. J Bioenerg Biomembr. 41 (6):493-497 (2009).
- 18. Marella M, Seo B B, Thomas B B, Matsuno-Yagi A, Yagi T. Successful amelioration of mitochondrial optic neuropathy using the yeast NDI1 gene in a rat animal model. PLoS One. 5 (7):e11472 (2010).
- 19. Palfi A, Millington-Ward S, Chadderton N, O'Reilly M, Goldmann T, Humphries M M, Li T, Wolfrum U, Humphries P, Kenna P F, Farrar G J. Adeno-associated virus-mediated rhodopsin replacement provides therapeutic benefit in mice with a targeted disruption of the rhodopsin gene. Hum Gene Ther. 2010 March; 21 (3):311-23.
- 20. Ayuso E, Mingozzi F, Montane J, Leon X, Anguela X M, Haurigot V, Edmonson S A, Africa L, Zhou S, High K A, Bosch F, Wright J F High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther. 2010 April; 17 (4):503-10.
- 21. Rohr U P, Wulf M A, Stahn S, Steidl U, Haas R, Kronenwett R. Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR. J Virol Methods. 2002 October; 106(1):81-8.
- 22. Kormann M S, Hasenpusch G, Aneja M K, Nica G, Flemmer A W, Herber-Jonat S, Huppmann M, Mays L E, Illenyi M, Schams A, Griese M, Bittmann I, Handgretinger R, Hartl D, Rosenecker J, Rudolph C. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 2011 February; 29 (2):154-7. doi: 10.1038/nbt.1733. Epub 2011 Jan. 9.
- 23. Chaput J C, Yu H, Zhang S. The emerging world of synthetic genetics. Chem. Biol. 2012 Nov. 21; 19 (11):1360-71. doi: 10.1016/j.chembiol.2012.10.011.
