Patent Abstract:
An isolated nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a functional variant thereof is described. The nucleic acid sequence comprises at least 50 codons which are codon optimised compared with the sequence of yeast NDI1 gene of SEQ ID NO: 1.

Full Description:
TECHNICAL FIELD 
       [0001]    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. 
       INTRODUCTION 
       [0002]    Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder affecting 1/25,000 people, predominantly males 1 . Loss of central vision results from the degeneration of the retinal ganglion cell (RGC) layer and optic nerve 2 . In over 95% of patients the genetic pathogenesis of LHON involves mutations in genes encoding components of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex 3  (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 (http://www.mitomap.org/MITOMAP). 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 glaucoma 4 . 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 supply 6 . 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 phosphorylation 7  (OXPHOS). Mitochondrial complex I mutations leading to respiratory chain dysfunction are hence linked to reduced oxygen consumption; a reliable measure of overall mitochondrial activity. 
         [0003]    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 function 8 . 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 production 9 . 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 loss 10 . Nuclear modifier genes can influence LHON progression and severity, for example, an x-linked modifier locus has been reported 11 . Additionally, smoking has been suggested as one of the environmental factors which can influence disease penetrance 12 . In addition, the male prevalence (5:1) of LHON may at last in part be influenced by oestrogens 13 . 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. 
         [0004]    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 mitochondria 14,15,16 . 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 readouts 18 . 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. 
         [0005]    It is an object of the invention to overcome at least one of the above-referenced problems. 
       STATEMENTS OF INVENTION 
       [0006]    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: 
         [0007]    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: 
         [0008]    L195F, K284E, K10R, S143N, L502M, L4031, 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. 
         [0009]    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. 
         [0010]    Examples of codon optimised variants of yeast NDI1 gene are provided in SEQ ID NO&#39;S: 2-62, 75-145, 165-243, 264-341, 362-441, 462-541, and 705-1004. 
         [0011]    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: 
         [0012]    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: 
         [0013]    L195F, K284E, K10R, S143N, L502M, L4031, 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. 
         [0014]    Examples of immune and codon optimised variants of yeast NDI1 gene are provided in SEQ ID NO&#39;S: 75-145, 165-243, 264-341, 362-441, 462-541, 566-584, 705-824, 835-884, 895-944 and 955-1004. 
         [0015]    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: 
         [0016]    L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259, K511, L159, R479, L483, 182, 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: 
         [0017]    L195F, K284E, K10R, S143N, L502M, L4031, 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, 
         [0018]    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. 
         [0019]    Examples of immune optimised variants of yeast NDI1 gene (without codon optimisation) are provided in SEQ ID NO&#39;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). 
         [0020]    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. 
         [0021]    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, L4031, 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. 
         [0022]    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. 
         [0023]    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, L4031, 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. 
         [0024]    Ideally, the variant protein includes an amino acid change selected from: L195F, K284E, K10R, S143N, L502M, L4031, 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. 
         [0025]    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&#39;s 62, 134-145, 225-243, 324-341, 422-441, 522-541, 566-584 and 705-824). 
         [0026]    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. 
         [0027]    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. 
         [0028]    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). 
         [0029]    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. 
         [0030]    In one aspect the invention relates to delivery of RNA encoding the protein and or protein variants of the invention. 
         [0031]    The invention also relates to a protein encoded by a nucleic acid construct of the invention. 
         [0032]    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. 
         [0033]    The invention also relates to a nucleic acid sequence selected from SEQ ID NO&#39;s: 1-541 and 547-1004. 
         [0034]    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). 
         [0035]    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 vechicles, 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. 
         [0036]    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. 
         [0037]    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. 
         [0038]    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&#39;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&#39;s. 
         [0039]    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. 
         [0040]    In an additional aspect, the nucleic acid of the invention is expressed from an inducible and/or conditional promotor. 
         [0041]    In a further embodiment, the promotor is a tissue specific and/or cell specific promotor targeting mammalian cells other than RGC&#39;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. 
         [0042]    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. 
         [0043]    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. 
         [0044]    Preferably, the step of delivering the nucleic acid of the invention involves delivering a vector of the invention to the individual. 
         [0045]    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. 
         [0046]    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. 
         [0047]    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. 
         [0048]    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. 
         [0049]    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&#39;s disease, Parkinson&#39;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. 
         [0050]    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. 
         [0051]    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. 
         [0052]    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. 
         [0053]    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. 
         [0054]    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). 
         [0055]    In an additional embodiment encapsulated cell technology is employed for delivery of the therapy. 
         [0056]    In one embodiment the invention provides a transgenic organ, or a transgenic non-human animal, comprising the nucleic acids and vectors of the invention. 
         [0057]    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. 
         [0058]    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. 
         [0059]    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. 
         [0060]    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. 
         [0061]    The term “yeast NDI1 gene” refers to the wild-type  Saccharomyces cerviscae  NDI1 gene shown in SEQ ID NO: 1. 
         [0062]    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. 
         [0063]    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. 
         [0064]    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. 
         [0065]    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. 
         [0066]    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. 
         [0067]    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/ FIG. 2 ). Typically, the functional variants of yeast NDI1 protein will have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 542. In this context, a polypeptide sequence that shares 90% amino acid identity with SEQ ID NO: 542 is one in which any 90% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in SEQ ID NO: 542. The “percent sequence identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul  Proc. Natl. Acad. Sci . USA 87:2264-68, 1990, modified as in Karlin and Altschul  Proc. Natl. Acad. Sci . USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.  J. Mol. Biol.  215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al.,  Nucleic Acids Res.  25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. 
         [0068]    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&#39;s disease; Parkinson&#39;s disease; Parkinson&#39;s plus; Tauopathies; Chromosome 17 dementias; Alzheimer&#39;s disease; Multiple sclerosis (MS); hereditary and acquired neuropathies; retinopathies and diseases involving cerebellar degeneration. 
         [0069]    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&#39;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&#39; cells. Other types of vectors suitable for use in gene therapy are described below. 
         [0070]    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. 
         [0071]    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. 
         [0072]    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. 
         [0073]    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). 
         [0074]    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. 
         [0075]    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. 
         [0076]    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. 
         [0077]    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.) 
         [0078]    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)). 
         [0079]    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. 
         [0080]    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&#39;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. 
         [0081]    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. 
         [0082]    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&#39;s circumstances. 
       Nucleic Acid Sequences of the Invention 
       [0083]    The sequence listing below provides a number of nucleic acid sequences according to the invention, specifically: 
         [0084]    SEQ ID NO: 1—Yeast NDI1 gene—0 amino acid changes—0 codon changes 
         [0085]    SEQ ID NO&#39;S 2-21 and 825-834—Yeast NDI1 gene—0 amino acid changes—100 codon changes 
         [0086]    SEQ ID NO&#39;S 22-41 and 885-894—Yeast NDI1 gene—0 amino acid changes—200 codon changes 
         [0087]    SEQ ID NO&#39;S 42-61 and 945-954—Yeast NDI1 gene—0 amino acid changes—300 codon changes 
         [0088]    SEQ ID NO 62—Yeast NDI1 gene—0 amino acid changes—329 codon changes 
         [0089]    SEQ ID NO&#39;S 63-74 and 547-565—Yeast NDI1 gene—1 amino acid changes—0 codon changes 
         [0090]    SEQ ID NO&#39;S 75-94 and 835-844—Yeast NDI1 gene—1 amino acid changes—100 codon changes 
         [0091]    SEQ ID NO&#39;S 95-114 and 895-904—Yeast NDI1 gene—1 amino acid changes—200 codon changes 
         [0092]    SEQ ID NO&#39;S 115-134 and 955-964—Yeast NDI1 gene—1 amino acid changes—300 codon changes 
         [0093]    SEQ ID NO&#39;S 134-145 and 566-584—Yeast NDI1 gene—1 amino acid changes—329 codon changes 
         [0094]    SEQ ID NO&#39;S 146-164 and 585-605—Yeast NDI1 gene—2 amino acid changes—0 codon changes 
         [0095]    SEQ ID NO&#39;S 165-184 and 845-854—Yeast NDI1 gene—2 amino acid changes—100 codon changes 
         [0096]    SEQ ID NO&#39;S 185-204 and 905-914—Yeast NDI1 gene—2 amino acid changes—200 codon changes 
         [0097]    SEQ ID NO&#39;S 205-224 and 965-974—Yeast NDI1 gene—2 amino acid changes—300 codon changes 
         [0098]    SEQ ID NO&#39;S 225-243 and 705-725—Yeast NDI1 gene—2 amino acid changes—329 codon changes 
         [0099]    SEQ ID NO&#39;S 244-263 and 606-640—Yeast NDI1 gene—3 amino acid changes—0 codon changes 
         [0100]    SEQ ID NO&#39;S 264-283 and 855-864—Yeast NDI1 gene—3 amino acid changes—100 codon changes 
         [0101]    SEQ ID NO&#39;S 284-303 and 915-924—Yeast NDI1 gene—3 amino acid changes—200 codon changes 
         [0102]    SEQ ID NO&#39;S 304-323 and 975-984—Yeast NDI1 gene—3 amino acid changes—300 codon changes 
         [0103]    SEQ ID NO&#39;S 324-341 and 726-760—Yeast NDI1 gene—3 amino acid changes—329 codon changes 
         [0104]    SEQ ID NO&#39;S 641-675—Yeast NDI1 gene—4 amino acid changes—0 codon changes 
         [0105]    SEQ ID NO&#39;S 865-874—Yeast NDI1 gene—4 amino acid changes—100 codon changes 
         [0106]    SEQ ID NO&#39;S 925-934—Yeast NDI1 gene—4 amino acid changes—200 codon changes 
         [0107]    SEQ ID NO&#39;S 985-994—Yeast NDI1 gene—4 amino acid changes—300 codon changes 
         [0108]    SEQ ID NO&#39;S 761-795—Yeast NDI1 gene—4 amino acid changes—329 codon changes 
         [0109]    SEQ ID NO&#39;S 342-361 and 676-696—Yeast NDI1 gene—5 amino acid changes—0 codon changes 
         [0110]    SEQ ID NO&#39;S 362-381 and 875-884—Yeast NDI1 gene—5 amino acid changes—100 codon changes 
         [0111]    SEQ ID NO&#39;S 382-401 and 935-944—Yeast NDI1 gene—5 amino acid changes—200 codon changes 
         [0112]    SEQ ID NO&#39;S 402-421 and 995-1004—Yeast NDI1 gene—5 amino acid changes—300 codon changes 
         [0113]    SEQ ID NO&#39;S 422-441 and 796-816—Yeast NDI1 gene—5 amino acid changes—329 codon changes 
         [0114]    SEQ ID NO&#39;S 697-703—Yeast NDI1 gene—6 amino acid changes—0 codon changes 
         [0115]    SEQ ID NO&#39;S 817-823—Yeast NDI1 gene—6 amino acid changes—329 codon changes 
         [0116]    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 
         [0117]    SEQ ID NO&#39;S 442-461—Yeast NDI1 gene—10 amino acid changes—0 codon changes 
         [0118]    SEQ ID NO&#39;S 462-481—Yeast NDI1 gene—10 amino acid changes—100 codon changes 
         [0119]    SEQ ID NO&#39;S 482-501—Yeast NDI1 gene—10 amino acid changes—200 codon changes 
         [0120]    SEQ ID NO&#39;S 502-521—Yeast NDI1 gene—10 amino acid changes—300 codon changes 
         [0121]    SEQ ID NO&#39;S 522-541—Yeast NDI1 gene—10 amino acid changes—329 codon changes 
         [0122]    SEQ ID NO: 542—Yeast NDI1 protein—0 amino acid changes 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0123]      FIG. 1 . Diagrammatic representation of the core construct designs. A: OphNDI1; OphNDI1 (yeast NDI1 gene which has been codon optimized and/or immune optimized) was expressed from the CMV (cytomegalovirus) immediate early promoter. A minimal polyadenylation signal was located at the 3′ end of the NDI1 gene. B: AAV-GDNF; GDNF (glial cell line derived neurotrophic factor) was expressed from the short ubiquitin promoter. The neurturin polyadenylation signal was located at the 3′ end of the GDNF gene. C: AAV-OphNDI1_GDNF; OphNDI1 was expressed from the CMV immediate early promoter. A minimal polyadenylation signal was located at the 3′ end of the NDI1 gene. 3′ to this GDNF was expressed from the short ubiquitin promoter. The neurturin polyadenylation signal was located at the 3′ end of the GDNF gene. D: OphNdiI expressed from a CMV promoter with a 3′ minimal polyadenylation signal. In this construct GDNF is expressed from an IRES and also contains the neurturin Polyadenylation signal. 
           [0124]    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 &amp; 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 &amp; 1b and the attached Sequence Listing. Notably, different polyadenalation signals may also be utilised in the constructs described. 
           [0125]      FIG. 2 . Localisation, function and mRNA expression of NDI1. Western blot analysis of mitochondrial protein isolated from pAAV-NDI1 transfected and untransfected (Ctrl) HeLa cells (A). Top panel shows NDI1 protein expression (56 KDa) and bottom panel shows VDAC1 protein expression (31 KDa, mitochondrial loading control; n=3). B. Bar chart represents oxygen consumption measurements from pAAV-NDI1 transfected (black columns) and pAAV-EGFP transfected (Ctrl, white columns) HeLa cells with (+) and without (no) 5 μmol rotenone (n=6). C. Bar chart represents percentage rotenone insensitive respiration in pAAV-NDI1 transfected (black columns) and pAAV-EGFP transfected (control, white columns) HeLa cells (n=6). D. Retinal NDI1 mRNA expression from adult wild type mice intravitreally injected with 3×10 8  vp AAV-NDI1 or 3×10 8  vp AAV-EGFP (Ctrl) and analysed by RT-PCR two weeks post-injection (n=6). Rot insensitive resp (%): Percentage rotenone insensitive respiration, w: water blank, M: size marker; KDa (A), by (D). Error bars represent SD values and *: p&lt;0.001. 
           [0126]      FIG. 3 . Oxygen consumption measurements from NDI1 transfected HeLa cells. Oxygen consumption measurements from HeLa cells transfected with pAAV-NDI1 (A) and pAAV-EGFP (B) in the presence of 5 μmol rotenone. Oxygen consumption measurements from HeLa cells transfected with pAAV-NDI1 (C) and pAAV-EGFP (D) in the absence of rotenone (control). 
           [0127]      FIG. 4   a . Oxygraphs for NDI1 constructs. Traces showing oxygen concentration (blue line) and oxygen consumption (red line) in media treated with 5 μmol rotenone and untransfected HeLa cells (negative control, A), cells transfected with ophNDI1-I82V (B), containing codon-optimisation at 329 codons and the I82V substitution and cells transfected with NDI1-I82V (C). Representative graphs for each are presented. Similarly HeLa cells were transfected with V45I constructs either the codon optimised hNDI1-V45I construct (D) or the wild type NDI1 construct containing the V45I substitution (NDI1-V45I; E). In addition V266I constructs, both NDI1-V266I (F) and hNDI1-V266I (G) were evaluated. The NDI1-F90Y (H) and hNDI1-F90Y (I) construct was also tested in HeLa cells treated with rotenone. 
           [0128]      FIG. 4   b . Bar charts of the data sets measuring the change in oxygen consumption from the experiments in  FIG. 4   a  are presented. A statistically significant retention in oxygen consumption was observed between cells transfected with either the NDI1 variant or the hNDI1 variant constructs with p values ranging from p&lt;0.05 (*) to &lt;0.01 (**). A significant difference was observed between the rotenone insensitive respiration achieved with I82V and V45I constructs versus that achieved with the F90Y construct (I82V versus F90Y p&lt;0.02 and V45I versus Y90Y p&lt;0.002). No significant differences were observed between NDI1 treated cells and cells treated with NDI1-I82V, hNDI1-I82V or V45I constructs. However F90Y transfected cells differed significantly compared to NDI1 transfected cells, the latter showing a better retention of oxygen consumption. 
           [0129]      FIG. 5 . Histology of NDI1 treated retinas following rotenone insult. Adult wild type mice were intravitreally injected into contralateral eyes with 3×10 8  vp AAV-NDI1 (A) and 1×10 8  vp AAV-EGFP, to facilitate localisation of transduced regions of the retinas, or 3×10 8  vp AAV-EGFP (B) alone (n=4). Three weeks post-injection, 1.5 nmol of rotenone was administered intravitrally to both eyes. Three weeks post-rotenone treatment eyes were enucleated, fixed, cryosectioned (12 μm) and processed for immunocytochemistry using NeuN primary and Cy3-conjugated secondary antibodies. Nuclei were counterstained with DAPI. A and B: representative sections show NeuN labelling (red) and nuclear DAPI (blue) signals overlaid. OS: photoreceptor outer segments; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bar: 20 μm. C: Bar chart representing mean ganglion cell counts per 100 μm. Blue and white columns represent values corresponding to AAV-NDI1+rotenone (NDI1) and AAV-EGFP+rotenone (EGFP), respectively. Error bars represent SD values and ***: p&lt;0.001. 
           [0130]      FIG. 6 . Ultra-structural analysis of NDI1 treated optic nerves following rotenone insult. Adult wild type mice were intravitrally injected into contralateral eyes with AAV-NDI1 (B) or AAV-EGFP (C and D) (n=3). Three weeks post-injection, 1.5 nmol of rotenone was administered intravitreally to both eyes. Three weeks later eyes were enucleated and optic nerves collected, post-fixed, processed and analysed by transmission electron microscopy. At low magnification electron dense structures (arrow heads, B and C) were less frequent in the AAV-NDI1+rotenone (B) treated samples compared to the AAV-EGFP+rotenone treated samples (C). AAV-EGFP+rotenone treated samples at higher magnification (D). These were not apparent in the untreated samples (A). E: Bar chart representing mean number of membrane debris. Black and white columns represent AAV-NDI1+rotenone (NDI1) and AAV-EGFP+rotenone (EGFP), respectively. F: Bar chart representing mean optic nerve diameter measurements. Optic nerves from identically injected mice were taken nine months post-rotenone treatment, fixed, cryosectioned (12 μm) and the thickness of the optic nerve measured using light microscopy. Black and white columns represent AAV-NDI1+rotenone (NDI1) and AAV-EGFP+rotenone (EGFP), respectively. Error bars represent SD values and **: p&lt;0.01. Scale bars: 10 μm (A, B and C) and 2 μm (D). 
           [0131]      FIG. 7 . Functional analysis of AAV-NDI1 and AAV-NSG treated optic nerves following rotenone insult. Adult wild type mice were intravitreally injected into the right eye with AAV-NDI1 (n=10) or AAV-NSG (n=6). Three weeks later, AAV-NDI1 (n=10) or AAV-NSG (n=6) injected mice received 1.5 nmol rotenone in the right eye. A further group of adult wild type mice received either DMSO (vehicle control, n=16) or 1.5 nmol rotenone intravitreally injected into the right eye (n=16). Two weeks post rotenone, or DMSO, treatment each mouse was intravitreally injected with 40 μg manganese chloride and manganese enhanced magnetic resonance imaging (MEMRI) carried out 2 hrs later. Pseudo-coloured T1-weighted images: Signal enhancement of the mouse visual pathway in oblique sections (36°) from DMSO (A), rotenone alone (B), AAV-NDI1+rotenone (C) and AAV-NSG+rotenone (D) are presented. E: Bar chart representing mean lg signal intensities in the region of the optic chiasm calculated using Image J® software. a.u.: arbitrary unit. Error bars represent SD values and ** represent p&lt;0.01. 
           [0132]      FIG. 8 . Analysis of spatial vision in NDI treated mice following rotenone insult. Adult wild type mice were intravitrally injected into contralateral eyes with 3×10 9  vp AAV-NDI1 or 3×10 9  vp AAV-EGFP. Three weeks post-injection, 1.5 nmol of rotenone was administered intravitreally to both eyes; control mice were not administered with rotenone. Three months post-rotenone treatment optokinetic responses were measured using a virtual optokinetic system. Bar chart represents the mean spatial frequency threshold established for each eye. Black and white columns represent values corresponding to AAV-NDI1+rotenone (NDI1) and AAV-EGFP+rotenone (EGFP), respectively in rotenone treated (+Rotenone) and control (No Rotenone) mice. Error bars represent SD values and ***: p&lt;0.001. 
           [0133]      FIG. 9   a . A representative western blot of proteins extracted from HeLa cells transiently transfected with plasmids expressing OphNDI1 and NDI1. A polyclonal antibody for Ndi1 was used to detect OphNDI1 and Ndi1 protein expressed in transfected cells. Lane 1; Ndi1 protein expressed from the original wild type NDI1 construct, Lane 2; Ndi1 with a C-terminal HA tag, Lane 3; Ndi1 protein expressed from OphNDI1, a humanized NDI1 construct with 329 optimised codons, Lane 4; Ndi1 protein expressed from OphNDI1-HA, a humanized Ndi1 with a HA tag. Lane 5; untransfected HeLa cells. 
           [0134]      FIG. 9   b : Bar chart showing normalized expression of humanized and wild-type Ndi1 protein as measured by western blot. HeLa cells were transfected with humanized and wild-type Ndi1. Cells were harvested 48 hours post-transfection and protein was extracted and western blotted using a polyclonal anti-Ndi1 primary antibody. Four independent blots were performed and images were captured and analysed with ImageJ® software to measure relative expression. For each blot, the relative expression level of wild-type Ndi1 was taken as a reference and the expression level of humanized Ndi1 was directly compared to it. Paired t-test performed on the non-normalized values indicate that humanized Ndi1 expresses significantly more highly than wild-typeNdI1 (P&lt;0.005). a.u.:arbitrary unit 
           [0135]      FIG. 10 . Expression from AAV vectors expressing variants of NDI1 AAV vectors were intravitreally injected into wild type mice. AAV vectors contained unmodified NDI1, NSG (expressing both unmodified NDI1 and a GDNF gene), modified NDI1 with a V266I modification, humanised NDI1 (hNDI1), or hNDI1 with a I82V modification. Two weeks post-injection retinas were harvested and total RNA extracted. Real time RT PCRs were performed on RNA samples using primers NDI1F and NDI1R and hNDI1F and hNDI1 R. 
           [0136]    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. 
           [0137]    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  FIGS. 5A and 5B  are expressed in copy number per unit of the housekeeping gene β-actin, expression levels may be compared directly. 
           [0138]    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  FIGS. 10   a  and  10   b.    
           [0139]      FIG. 11 . Immunogenicity predictions of each 9-mer peptide fragment in NDI1, via in silico modelling of antigen presentation using the MHC class I predictor alone ( FIG. 11   a ) or employing the MHC-I pathway using the IEDB proteasomal cleavage/TAP transport/MHC class I combined predictor ( FIG. 11   b ). Immunogenicity scores and amino acid positions are presented. 
           [0140]      FIG. 12A . Oxygraphs for NSG constructs Trace showing oxygen concentration (blue line) and oxygen consumption (red line) in media containing untransfected cells (negative control A), cells transfected with wild-type Ndi1 (B) and cells transfected with NSG, a construct expressing both wild-type NDI1 and GDNF (C). In each case, cells were analysed without rotenone and a steady respiration level measured. Once respiration stabilized and a measurement taken, 5 μmol rotenone was added and a measurement of rotenone-insensitive respiration taken once oxygen consumption stabilized. 
           [0141]      FIG. 12B : A bar chart of the data from NSG and NDI1 transfected HeLa cells is presented. NSG and NDI1 transfected HeLa cells did not differ significantly from each other p=0.6, however, both significantly retained oxygen consumption compared to untransfected controls (NSG p&lt;0.05 and NDI1 p&lt;0.01). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0142]    In the present invention delivery of NDI1 constructs ( FIG. 1 ) has been used to protect cells in the presence of a complex I inhibitor, rotenone, ( FIGS. 2-8  and  12 ), HeLa cells and retinal ganglion cells (RGCs) were protected in the presence of NDI1 delivered as a wild type construct or as codon-optimised and immuno-optimised constructs ( FIGS. 2-4 ). For example, RGCs, the cells primarily affected in LHON, were protected in a rotenone-induced murine model of LHON. Recombinant AAV serotype 2 (AAV2/2) expressing wild type NDI1 from a CMV promoter (AAV-NDI1,  FIG. 1A ) was administered to mice using a single intravitreal injection. AAV2/2 administered through this route has been shown to infect RGCs efficiently. Moreover, intravitreal injection typically results in a broad area of retinal transduction as the vitreous contacts the entire underlying retinal surface 32 . Intravitreal injection of AAV provides a route of administration for the gene therapy which is directly applicable to human patients and is routinely used to administer drugs such as Avastin and Lucentis for treatment of wet AMD. In this study, intravitreal injection of AAV-NDI1 was utilised for the first time and was shown significantly to reduce RGC death and optic nerve atrophy seen in untreated eyes in response to rotenone administration and moreover, led to a preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging (MEMRI) and optokinetic responses (OKR;  FIGS. 5-8 ). 
         [0143]    In the present Application, intravitreal injection of AAV-NDI1 provided substantial protection against rotenone-induced insult, as assessed by a variety of assays ( FIGS. 5-8 ). Notably, histological analyses demonstrated significant protection of both RGCs and the optic nerve ( FIGS. 5 and 6 ). Furthermore, MEMRI indicated that AAV-NDI1 treatment preserved optic nerve function by enabling active transport of manganese ions through the optic nerve using voltage-gated calcium channels and hence provided evidence of the improved functional integrity of the optic nerve tissue in AAV-NDI1 treated eyes compared to control eyes ( FIG. 7 ). Evaluation of visual function by optokinetics showed that the protection of RGCs and optic nerve integrity afforded by AAV-NDI1 led to preservation of mouse vision in the presence of the complex I inhibitor rotenone ( FIG. 8 ). The results highlight the potential therapeutic value of NDI1-based therapies for LHON when intravitreally delivered using AAV2/2. 
         [0144]    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 ( FIGS. 9   a ,  9   b  and  10 ). A statistically significant difference in levels of expression was obtained between wild type and optimized NDI1 constructs ( FIGS. 9   a  and  9   b ). The results obtained for NDI1 protein ( FIGS. 9   a  and  9   b ) are mirrored by those obtained at the RNA level in mice intravitreally injected with AAV wild type and optimized NDI1 constructs using real-time RT PCR as the assay ( FIG. 10 ). 
         [0145]    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 &amp; 1b and Sequence Listing). Amino acid modifications were undertaken subsequent to in silico analyses for potential immunogenic sites within NDI1 (see  FIGS. 11   a  and  11   b , material and methods). Immuno-optimised constructs were generated for both the wild type NDI1 construct and for the codon-optimised hNDI1 construct. Modified codon-optimised and immuno-optimised NDI1 constructs were generated as high titre AAV2/2 vectors (1-5×10 11  vg/ml) using triple plasmid transfection methods in 293 cells followed by cesium chloride gradient purification of virus. Representative immuno-optimised NDI1 and immuno-optimised hNDI1 constructs inter alia V45I, I82V, L89I, I90Y, V266I, L481I, L483M were generated as plasmids and or AAV vectors. All nucleated mammalian cells present peptide fragments bound to MHC-I molecules on their cell surface. These fragments are derived from the degradation of proteins in the cytoplasm. As such, MHC-I presentation offers a snapshot of the pool of proteins being produced within each cell. Cytotoxic T-cells inspect the peptide fragments presented by cells and can induce apoptosis in cells presenting non-self proteins, which is usually an indicator of viral infection. HeLa cells were transfected with NDI1, hNDI1 and immuno-optimised constructs and levels of rotenone insensitive respiration evaluated ( FIGS. 2-4 ,  12 ). Significant retention of oxygen consumption was observed in cells transfected with NDI1, codon-optimised and immuno-optimised constructs ( FIGS. 2-4 ,  12 ), when compared to untransfected control cells. 
         [0146]    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 ( FIG. 1 ) and generated as an AAV2/2 vector (AAV-NSG). Significantly higher levels of expression of NDI1 were achieved from this vector in vivo in mice after intravitreal injection compared to AAV-NDI1 as evaluated by real time RT-PCR assays ( FIGS. 10   a  and  10   c ). GDNF expression from AAV-NSG was confirmed in mouse retinas by real time RT-PCR. Furthermore, intravitreally delivery of AAV-NSG resulted in preservation of cell function as evaluated by oxygen consumption measurements in rotenone treated HeLa cells ( FIG. 12 ) and functional preservation in vivo using MRI analyses of wild type mice intravitreally injected with AAV-NSG vector ( FIG. 7 ). Mean MRI signal intensity for DMSO was 2.38±0.04, for rotenone alone was 2.30±0.06, for AAV-NDI1 plus rotenone was 2.35±0.07 and for AAV-NSG plus rotenone was 2.37±0.07, significant differences were found between the rotenone alone treated mice and those treated with rotenone and either AAV-NDI1 or AAV-NSG; for both rotenone versus AAV-NDI1 and rotenone versus AAV-NSG comparisons, p&lt;0.01 (**). Indeed AAV-NDI1 (plus rotenone) or AAV-NSG (plus rotenone) treated mice did not differ significantly from wild type control mice treated with DMSO alone. Notably these MRI results were established using a 4-fold lower titre of AAV-NSG than AAV-NDI1 (5.99×10 11  vp/ml versus 2.5×10 11  vp/ml) Suggesting that less AAV-NSG is required to mediate an equivalent beneficial effect. 
         [0147]    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 ( FIG. 10 ). Levels of expression from different vectors could be directly compared as expression was evaluated by absolute copy number per unit of β-actin (the housekeeping control) for each vector. The standard curves were generated using plasmid DNA standards with known copy number. Expression levels achieved after AAV intravitreal injection of vectors were greater in mouse eyes treated with AAV-hNDI1 or AAV-NSG treated eyes compared to AAV-NDI1 injected eyes ( FIG. 10 ). 
         [0148]    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. 
         [0149]    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. 
         [0150]    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. 
         [0151]    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 
       [0152]    Vector Construction and AAV Production 
         [0153]    Yeast NDI1 (Accession No: NM — 001182483.1) was cloned as described 53 . 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 signals 4  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;  FIG. 1 . pAAV-EGFP was cloned as previously described 19 . 
         [0154]    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). 
         [0155]    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. 
         [0156]    Recombinant AAV2/2 viruses, AAV-ND1, AAV-NSG, pAAV-NDI1 V266I, AAV-huNDI1, pAAV-huNDI1 182V and AAV-EGFP were prepared as described 20 , with a modified cesium chloride gradient as described 19  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 
       [0157]    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&#39;s instructions (Invitrogen, Carlsbad, Calif., USA). 5×10 5  cells per well were seeded onto 6-well plates containing 1 ml Dulbecco&#39;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 
       [0158]    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 homogenised 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 cocktail tablet/10 mls (Roche, Mannheim, Germany)). The homogenate 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 
       [0159]    Respiratory measurements were performed in DMEM at 37° C. on an Oxygraph-2k (OROBOROS® INSTRUMENTS GmbH, Innsbruck, Austria) according to the manufacturer&#39;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 1×10 6  pAAV-NDI1 or 1×10 6  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 1×10 6  pAAV-NDI1 or 1×10 6  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 
       [0160]    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 described 57 . 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 1×10 12  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&#39; Institutional Review Board. 
       RNA Extraction and PCR Analysis 
       [0161]    Adult wild type mice (n=6) were intravitrally injected with 3×10 9  vp AAV-NDI1 while fellow eyes received 3×10 9  vp AAV-EGFP. Retinas were harvested two weeks post-injection and total RNA extracted using the Qiagen RNeasy kit according to the manufacturers 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) using a QuantiTect SYBR Green RT-PCR kit (Qiagen Ltd., Crawley, UK) 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′ and hNDI1 reverse primer 5′ GCTGATCAGGTAGTCGTACT 3′ β-actin was used as an internal control as described (ref). RT-PCRs were performed twice in triplicate or quadruplicate. Levels of NDI1 or humanised 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 
       [0162]    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 
       [0163]    Optic nerve integrity in experimental and control mice was assessed by Manganese (Mn 2+ ) 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 Mn 2+  ions to enter excitable cells through voltage-gated calcium channels, thus analysis of Mn 2+  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&#39;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 T 1 -weighted 2D images were acquired using FLASH sequence (TR/TE:150/2.5 ms; Matrix: 128×128; Field of View: 20×20 mm 2 ; 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 (http://imagej.nih.gov/ij/). 
       Optokinetics 
       [0164]    Optokinetic response (OKR) spatial frequency thresholds were measured blind by two independent researchers using a virtual optokinetic system (VOS, OptoMotry, CerebralMechanics, Lethbridge, AB, Canada). OptoMotry 36  measures the threshold of the mouse&#39;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&#39;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 
       [0165]    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&#39;s two-tailed t-test or ANOVA used with Tukey&#39;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&lt;0.05 were considered statistically significant 
       Predictions of Immunogenic Codons 
       [0166]    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&lt;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. 
         [0167]    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. 
         [0168]    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 G p,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. 
         [0169]    An overall immunogenicity factor F p  for the 9-mer peptide was then calculated as 
         [0000]    
       
         
           
             
               F 
               p 
             
             = 
             
               
                 ∑ 
                 i 
               
                
               
                 
                   G 
                   
                     p 
                     , 
                     i 
                   
                 
                  
                 
                   N 
                   i 
                 
               
             
           
         
       
     
         [0000]    where N i  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, F p  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. 
         [0170]    Each amino acid position A in the Ndi1 peptide was then assigned an immunogenicity score S A  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. 
         [0171]    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 homologous protein sequences from many species against each other, and comparing the frequency with which each amino acid is replaced by every other amino acid. 
         [0172]    For two amino acids x and y, the BLOSUM score B x,y  is defined as the log-likelihood of the amino acid x replacing y or vice-versa in a given position in homologous peptides. As a direct consequence of this definition, B x,y =B y,x  for all x and y (in other words, all BLOSUM matrices are symmetric). 
         [0173]    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 homologous 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 (B x,x ), indicating the likelihood that that amino acid will remain constant between homologous proteins. 
         [0174]    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=B x,x −B x,y . All mutations for which ΔB was greater than 4 were considered too disruptive to protein function and not analysed further. 
         [0175]    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. 
         [0176]    All possible mutations were then ranked by the metric 
         [0000]    
       
         
           
             
               
                 Δ 
                  
                 
                     
                 
                  
                 S 
               
               
                 Δ 
                  
                 
                     
                 
                  
                 B 
               
             
             . 
           
         
       
     
         [0000]    High values of 
         [0000]    
       
         
           
             
               Δ 
                
               
                   
               
                
               S 
             
             
               Δ 
                
               
                   
               
                
               B 
             
           
         
       
     
         [0000]    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. 
         [0177]    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. 
       REFERENCES 
       [0000]    
       
         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&#39;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 3 rd . 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&#39;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, Miyashita K, Wakakura M, Oguchi Y. High incidence of visual recovery among four Japanese patients with Leber&#39;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&#39;Amati G, Carelli V. Oestrogens ameliorate mitochondrial dysfunction in Leber&#39;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&#39;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&#39;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&#39;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. 
       
     
       APPENDIX 
       [0201]      
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1a 
               
             
             
               
                   
               
               
                 Nucleic acid and amino acid sequences of the Invention. 
               
             
          
           
               
                   
                 Amino Acid  
                 Nucleic Acid  
               
               
                 Gene 
                 Substitution 
                 Sequence 
               
               
                   
               
               
                 Yeast NDI1 
                 FYLWRILYL 
                 SEQ ID NO: 1 
               
               
                   
               
               
                 Yeast NDI1 codon 
                 FYLWRILYL 
                 SEQ ID NO: 62 
               
               
                 optimised 
                   
                   
               
               
                   
               
               
                 Yeast NDI1 + 1amino 
                 FYLWRILYL → 
                 SEQ ID NO: 63 
               
               
                 acid change 
                 FYLWRILYM 
                   
               
               
                   
               
               
                 Yeast NDI1 codon 
                 FYLWRILYL → 
                 SEQ ID NO: 134 
               
               
                 optimized + 1 amino 
                 FYLWRILYM 
                   
               
               
                 acid change 
                   
                   
               
               
                   
               
               
                 Yeast NDI1 + 2 amino 
                 FYLWRILYL → 
                 SEQ ID NO: 146 
               
               
                 acid change 
                 FYLWRILYM 
                   
               
               
                   
                 FLKEIPNSL → 
                   
               
               
                   
                 FFKEIPNSL 
                   
               
               
                   
               
               
                 Yeast NDI1 codon 
                 FYLWRILYL → 
                 SEQ ID NO: 225 
               
               
                 optimized + 2 amino 
                 FYLWRILYM 
                   
               
               
                 acid change 
                 FLKEIPNSL → 
                   
               
               
                   
                 FFKEIPNSL 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1b 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Immunochange/ 
               
               
                 Initial  
                 Position 
                 New 
                 Immunoscore 
                 Immunochange 
                 Blosumchange 
                 Blosumchange 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 I 
                 82 
                 V 
                 2.569262982 
                 1.002693684 
                 2 
                 0.501346842 
               
               
                 F 
                 90 
                 Y 
                 1.926170105 
                 1.497108683 
                 3 
                 0.499036228 
               
               
                 L 
                 89 
                 I 
                 2.104411858 
                 1.253907982 
                 3 
                 0.417969327 
               
               
                 V 
                 266 
                 I 
                 0.667339713 
                 0.362552877 
                 1 
                 0.362552877 
               
               
                 K 
                 214 
                 E 
                 0.712950213 
                 0.70677809 
                 4 
                 0.176694523 
               
               
                 L 
                 481 
                 I 
                 0.885723713 
                 0.498012741 
                 3 
                 0.166004247 
               
               
                 L 
                 202 
                 M 
                 0.608956047 
                 0.315494717 
                 2 
                 0.157747359 
               
               
                 L 
                 259 
                 V 
                 0.594189679 
                 0.469145841 
                 3 
                 0.156381947 
               
               
                 L 
                 195 
                 I 
                 0.565666654 
                 0.465061673 
                 3 
                 0.155020558 
               
               
                 I 
                 81 
                 V 
                 0.852520887 
                 0.266903644 
                 2 
                 0.133451822 
               
               
                 L 
                 150 
                 M 
                 0.656551833 
                 0.259100799 
                 2 
                 0.129550399 
               
               
                 R 
                 85 
                 K 
                 2.714843954 
                 0.43039463 
                 4 
                 0.107598657 
               
               
                 Y 
                 151 
                 F 
                 0.686249712 
                 0.397772899 
                 4 
                 0.099443225 
               
               
                 Y 
                 482 
                 F 
                 0.891857027 
                 0.37332648 
                 4 
                 0.09333162 
               
               
                 S 
                 488 
                 T 
                 0.562058188 
                 0.361418691 
                 4 
                 0.090354673 
               
               
                 S 
                 80 
                 T 
                 0.674070843 
                 0.301172594 
                 4 
                 0.075293149 
               
               
                 K 
                 196 
                 E 
                 0.618739275 
                 0.284207587 
                 4 
                 0.071051897 
               
               
                 R 
                 206 
                 K 
                 0.780227471 
                 0.247789757 
                 4 
                 0.061947439 
               
               
                 R 
                 490 
                 K 
                 0.590906411 
                 0.237769694 
                 4 
                 0.059442424 
               
               
                 S 
                 145 
                 T 
                 0.67224222 
                 0.225480169 
                 4 
                 0.056370042 
               
               
                 V 
                 147 
                 T 
                 0.671708207 
                 0.210263616 
                 4 
                 0.052565904 
               
               
                 R 
                 479 
                 K 
                 1.226655337 
                 0.210156887 
                 4 
                 0.052539222 
               
               
                 A 
                 489 
                 S 
                 0.587738848 
                 0.201645996 
                 4 
                 0.050411499 
               
               
                 L 
                 212 
                 V 
                 0.717379457 
                 0.144498379 
                 3 
                 0.048166126 
               
               
                 R 
                 492 
                 K 
                 0.564269712 
                 0.191259766 
                 4 
                 0.047814941 
               
               
                 L 
                 262 
                 M 
                 0.596470347 
                 0.084255646 
                 2 
                 0.042127823 
               
               
                 Q 
                 149 
                 E 
                 0.656724126 
                 0.167775872 
                 4 
                 0.041943968 
               
               
                 T 
                 207 
                 S 
                 0.779275641 
                 0.162365948 
                 4 
                 0.040591487 
               
               
                 Y 
                 476 
                 F 
                 1.203763001 
                 0.154940174 
                 4 
                 0.038735043 
               
               
                 S 
                 201 
                 T 
                 0.598628015 
                 0.145693616 
                 4 
                 0.036423404 
               
               
                 S 
                 86 
                 A 
                 2.752011125 
                 0.111576956 
                 4 
                 0.027894239 
               
               
                 M 
                 473 
                 L 
                 0.621739886 
                 0.108503212 
                 4 
                 0.027125803 
               
               
                 E 
                 265 
                 Q 
                 0.583898093 
                 0.099401686 
                 4 
                 0.024850422 
               
               
                 E 
                 264 
                 Q 
                 0.583540076 
                 0.086415603 
                 4 
                 0.021603901 
               
               
                 S 
                 148 
                 A 
                 0.642943664 
                 0.069504199 
                 4 
                 0.01737605 
               
               
                 A 
                 261 
                 S 
                 0.592734437 
                 0.053926096 
                 4 
                 0.013481524 
               
               
                 A 
                 209 
                 S 
                 0.725497927 
                 0.039698254 
                 4 
                 0.009924564 
               
               
                 E 
                 213 
                 Q 
                 0.71301777 
                 0.004330404 
                 4 
                 0.001082601 
               
               
                   
               
               
                 Initial: The amino acid at this position in the native protein 
               
               
                 Position: Position in the protein 
               
               
                 New: Replacement amino acid suggested by the program 
               
               
                 Immunoscore: Immunoscore for this locus in the native protein. 
               
               
                 Immunochange: Change in immunoscore between the native and the modified locus. 
               
               
                 Blosumchange: The change in BLOSUM score between the native and the modified position (a measure of how conservative the change is, lower numbers being more conservative) 
               
               
                 Immunochange/Blosumchange: The change in immunogenicity divided by the blosum change. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1c 
               
             
             
               
                   
               
               
                 Output from immunogenicity analyses 
               
             
          
           
               
                 position 
                 totalscore 
                 mhcscore 
                 tapscore 
                 proteasomescore 
               
               
                   
               
             
          
           
               
                 0 
                 0.000165143 
                 11.14069809 
                 0.44351918 
                 9.06162E−05 
               
               
                 1 
                 0.041457346 
                 11.40019829 
                 2.86360034 
                 0.003448543 
               
               
                 2 
                 0.002426595 
                 15.73665433 
                 7.707479979 
                 5.46497E−05 
               
               
                 3 
                 0.002526801 
                 24.94091632 
                 4.435191796 
                 6.27721E−05 
               
               
                 4 
                 0.005897232 
                 16.1032081 
                 6.122268966 
                 0.000162811 
               
               
                 5 
                 0.00032745 
                 12.79123286 
                 1.221553255 
                 5.69915E−05 
               
               
                 6 
                 7.91604E−05 
                 15.37844434 
                 0.168621289 
                 8.22938E−05 
               
               
                 7 
                 0.000166722 
                 13.39406509 
                 0.702930528 
                 4.84144E−05 
               
               
                 8 
                 0.000109826 
                 14.68630033 
                 0.533227336 
                 3.79468E−05 
               
               
                 9 
                 0.000316701 
                 11.14069809 
                 1.308917895 
                 5.83262E−05 
               
               
                 10 
                 0.123430476 
                 22.74638603 
                 6.264874929 
                 0.002349837 
               
               
                 11 
                 0.097134418 
                 17.65681657 
                 2.552186489 
                 0.005899826 
               
               
                 12 
                 0.048628469 
                 20.27273789 
                 7.532036315 
                 0.000863136 
               
               
                 13 
                 0.046499396 
                 20.74495061 
                 21.22816259 
                 0.000286354 
               
               
                 14 
                 0.001693195 
                 20.74495061 
                 0.926642906 
                 0.000243003 
               
               
                 15 
                 2.94196E−05 
                 11.40019829 
                 0.150283882 
                 4.66216E−05 
               
               
                 16 
                 0.000555893 
                 11.14069809 
                 0.845108374 
                 0.000157342 
               
               
                 17 
                 0.062993668 
                 16.4783 
                 9.055499382 
                 0.001146233 
               
               
                 18 
                 0.000129314 
                 12.79123286 
                 0.212281626 
                 0.000129245 
               
               
                 19 
                 0.000372025 
                 11.93747308 
                 1.250006885 
                 6.79306E−05 
               
               
                 20 
                 0.000170764 
                 11.40019829 
                 0.656012939 
                 6.24617E−05 
               
               
                 21 
                 0.017125463 
                 13.39406509 
                 2.437319175 
                 0.00142792 
               
               
                 22 
                 8.29984E−05 
                 14.68630033 
                 0.360505924 
                 4.22373E−05 
               
               
                 23 
                 0.000159095 
                 14.02530793 
                 0.558357566 
                 5.49707E−05 
               
               
                 24 
                 3.85269E−05 
                 12.50006885 
                 0.222286151 
                 3.77629E−05 
               
               
                 25 
                 0.000126872 
                 14.68630033 
                 0.533227336 
                 4.48928E−05 
               
               
                 26 
                 0.000210364 
                 11.14069809 
                 0.970314241 
                 5.24778E−05 
               
               
                 27 
                 5.09001E−05 
                 9.929157627 
                 0.352299807 
                  4.009E−05 
               
               
                 28 
                 0.000168829 
                 8.070722319 
                 1.537844434 
                 3.71193E−05 
               
               
                 29 
                 0.000363844 
                 10.16043742 
                 0.671293443 
                 0.000146293 
               
               
                 30 
                 2.06189E−05 
                 11.66574302 
                 0.127912329 
                 3.82109E−05 
               
               
                 31 
                 0.022132896 
                 17.65681657 
                 16.1032081 
                 0.000209655 
               
               
                 32 
                 0.027879223 
                 8.84937105 
                 2.494091632 
                 0.003429894 
               
               
                 33 
                 0.000123359 
                 11.14069809 
                 0.641080261 
                 4.68999E−05 
               
               
                 34 
                 0.000806311 
                 20.74495061 
                 1.891961982 
                  5.6086E−05 
               
               
                 35 
                 0.000356829 
                 11.14069809 
                 1.806809666 
                 4.82049E−05 
               
               
                 36 
                 0.00055883 
                 18.4889567 
                 1.308917895 
                 6.21902E−05 
               
               
                 37 
                 0.00163762 
                 14.68630033 
                 2.734717094 
                 0.00010891 
               
               
                 38 
                 0.000115249 
                 22.22861507 
                 0.336443704 
                 4.12917E−05 
               
               
                 39 
                 0.000191343 
                 7.88701025 
                 1.765681657 
                 3.74729E−05 
               
               
                 40 
                 0.000491116 
                 6.712934432 
                 1.537844434 
                 0.000129807 
               
               
                 41 
                 0.000677471 
                 18.06809666 
                 1.166574302 
                 8.70739E−05 
               
               
                 42 
                 3.36856E−05 
                 11.14069809 
                 0.207449506 
                 3.88118E−05 
               
               
                 43 
                 0.00014483 
                 21.22816259 
                 0.377496044 
                 4.85281E−05 
               
               
                 44 
                 0.003264254 
                 7.360586237 
                 2.93030216 
                 0.000402442 
               
               
                 45 
                 0.000140007 
                 7.707479979 
                 1.402530793 
                  3.521E−05 
               
               
                 46 
                 3.62244E−05 
                 11.40019829 
                 0.232762174 
                 3.73826E−05 
               
               
                 47 
                 0.000348616 
                 16.1032081 
                 1.339406509 
                 4.33022E−05 
               
               
                 48 
                 0.004074612 
                 34.42804843 
                 3.774960444 
                 8.44971E−05 
               
               
                 49 
                 0.001131584 
                 11.66574302 
                 2.122816259 
                 0.000123023 
               
               
                 50 
                 0.002085286 
                 19.81127403 
                 3.213012427 
                 8.89479E−05 
               
               
                 51 
                 4.51837E−05 
                 10.39710441 
                 0.32878531 
                 3.62723E−05 
               
               
                 52 
                 3.03008E−05 
                 14.68630033 
                 0.114001983 
                 4.98593E−05 
               
               
                 53 
                 3.76548E−05 
                 10.88710484 
                 0.261163455 
                  3.596E−05 
               
               
                 54 
                 0.001659218 
                 2.79123286 
                 4.139161818 
                 8.50937E−05 
               
               
                 55 
                 0.000149148 
                 14.02530793 
                 0.545647796 
                 5.22381E−05 
               
               
                 56 
                 0.004171506 
                 12.50006885 
                 1.166574302 
                 0.000776349 
               
               
                 57 
                 0.006443566 
                 14.35199932 
                 2.611634549 
                 0.000465797 
               
               
                 58 
                 3.34281E−05 
                 14.68630033 
                 0.133940651 
                 4.56139E−05 
               
               
                 59 
                 0.037127736 
                 11.93747308 
                 16.1032081 
                 0.00052448 
               
               
                 60 
                 0.00663909 
                 19.81127403 
                 3.952868849 
                 0.000229639 
               
               
                 61 
                 0.006458684 
                 9.266429059 
                 2.494091632 
                 0.000758499 
               
               
                 62 
                 0.000340528 
                 20.27273789 
                 0.702930528 
                 6.40334E−05 
               
               
                 63 
                 0.002881924 
                 17.65681657 
                 2.494091632 
                 0.000178119 
               
               
                 64 
                 7.61656E−05 
                 14.68630033 
                 0.336443704 
                 4.10584E−05 
               
               
                 65 
                 0.000209237 
                 7.88701025 
                 1.140019829 
                 6.41994E−05 
               
               
                 66 
                 0.005967534 
                 9.703142406 
                 3.364437037 
                 0.000493477 
               
               
                 67 
                 0.001955737 
                 9.266429059 
                 2.552186489 
                 0.000221532 
               
               
                 68  
                 0.017604909 
                 20.27273789 
                 29.98557666 
                 7.89816E−05 
               
               
                 69 
                 8.35826E−05 
                 11.14069809 
                 0.558357566 
                 3.65261E−05 
               
               
                 70 
                 0.002594439 
                 19.36031438 
                 2.172263001 
                 0.000165466 
               
               
                 71 
                 3.00088E−05 
                 16.86212891 
                 0.122155325 
                 3.90383E−05 
               
               
                 72 
                 0.000919044 
                 11.66574302 
                 2.027273789 
                 0.000106306 
               
               
                 73 
                 0.000624075 
                 10.88710484 
                 2.494091632 
                 6.17683E−05 
               
               
                 74 
                 6.33881E−05 
                 11.40019829 
                 0.249409163 
                 6.13181E−05 
               
               
                 75 
                 0.000697186 
                 9.929157627 
                 2.672467333 
                 7.11732E−05 
               
               
                 76 
                 0.015140398 
                 10.39710441 
                 7.360586237 
                 0.000539173 
               
               
                 77 
                 6.53066E−05 
                 16.86212891 
                 0.19811274 
                 5.41402E−05 
               
               
                 78  
                 0.312040853 
                 26.72467333 
                 42.35575283 
                 0.000759428 
               
               
                 79 
                 0.344490583 
                 12.79123286 
                 27.98416838 
                 0.002643626 
               
               
                 80 
                 0.178480054 
                 26.11634549 
                 5.210895997 
                 0.003561048 
               
               
                 81 
                 1.717661138 
                 12.21553255 
                 21.72263001 
                 0.017177042 
               
               
                 82 
                 0.001404259 
                 20.74495061 
                 0.671293443 
                 0.000273623 
               
               
                 83 
                 0.000277344 
                 13.39406509 
                 1.468630033 
                 3.83701E−05 
               
               
                 84 
                 0.145284018 
                 12.50006885 
                 5.98290911 
                 0.005274918 
               
               
                 85 
                 0.052307569 
                 15.73665433 
                 7.532036315 
                 0.001198564 
               
               
                 86 
                 0.000858248 
                 9.482271919 
                 2.274638603 
                 0.000108393 
               
               
                 87 
                 0.007596331 
                 13.39406509 
                 0.172548984 
                 0.008925249 
               
               
                 88 
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                 89 
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                 1.686212891 
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                 90 
                 0.006680505 
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                 91 
                 6.22969E−05 
                 15.73665433 
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                 92 
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                 93 
                 0.011560202 
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                 94 
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                 95 
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                 96 
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                 97 
                 2.28667E−05 
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                  3.5317E−05 
               
               
                 98 
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                 99 
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                 100 
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                 101 
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                 102 
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                 103 
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                 104 
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                 105 
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                 106 
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                 107 
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                 108 
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                 109 
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                 110 
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                 111 
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                 112 
                 9.96662E−05 
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                 113 
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                 114 
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                 115 
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                 0.000583111 
               
               
                 116 
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                 117 
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                 118 
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                 119 
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                 120 
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                 121 
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                 122 
                 8.14247E−05 
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                 123 
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                 124 
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                 9.40827E−05 
               
               
                 125 
                 0.000141599 
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                  3.8882E−05 
               
               
                 126 
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                 7.09543E−05 
               
               
                 127 
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                 128 
                 7.74925E−05 
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                 129 
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                 130 
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                 0.000231783 
               
               
                 131 
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                 132 
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                 0.000226837 
               
               
                 133 
                 0.000243602 
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                 1.435199932 
                 3.90759E−05 
               
               
                 134 
                  2.0194E−05 
                 11.93747308 
                 0.122155325 
                 3.74976E−05 
               
               
                 135 
                 0.00240769 
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                 3.952868849 
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                 136 
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                 6.62302E−05 
               
               
                 137 
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                 9.76814E−05 
               
               
                 138 
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                 0.002447701 
               
               
                 139 
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                 140 
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                 4.46669E−05 
               
               
                 141 
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                 5.092281523 
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                 142 
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                 0.003851289 
               
               
                 143 
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                 7.55946E−05 
               
               
                 144 
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                 5.15599E−05 
               
               
                 145 
                 9.51453E−05 
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                 4.83954E−05 
               
               
                 146 
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                 4.28532E−05 
               
               
                 147 
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                 6.50063E−05 
               
               
                 148 
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                 5.713633843 
                 0.000249234 
               
               
                 149 
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                 0.000101384 
               
               
                 150 
                 0.072692591 
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                 4.644215946 
                 0.001979696 
               
               
                 151 
                 9.13848E−05 
                 8.451083744 
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                 4.90765E−05 
               
               
                 152 
                 0.000194321 
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                 4.05995E−05 
               
               
                 153 
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                 0.598290911 
                 4.97643E−05 
               
               
                 154 
                 5.96976E−05 
                 12.21553255 
                 0.313987533 
                  4.2448E−05 
               
               
                 155 
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                 1.64783 
                 4.72171E−05 
               
               
                 156 
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                 6.09429E−05 
               
               
                 157 
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                 9.055499382 
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                 4.26164E−05 
               
               
                 158 
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                 7.64658E−05 
               
               
                 159 
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                 2.734717094 
                 0.000166845 
               
               
                 160 
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                 0.000104775 
               
               
                 161 
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                 3.72481E−05 
               
               
                 162 
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                 21.72263001 
                 0.003405842 
               
               
                 163 
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                 4.12016E−05 
               
               
                 164 
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                 0.005646639 
               
               
                 165 
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                 4.45628E−05 
               
               
                 166 
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                 0.000205305 
               
               
                 167 
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                 168 
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                 169 
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                 4.99016E−05 
               
               
                 170 
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                 0.321301243 
                 0.000110242 
               
               
                 171 
                 0.000342173 
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                 1.981127403 
                  5.5619E−05 
               
               
                 172 
                 8.79781E−05 
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                 4.60152E−05 
               
               
                 173 
                 0.000211548 
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                 174 
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                 24.94091632 
                 0.00018851 
               
               
                 175 
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                 4.00369E−05 
               
               
                 176 
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                 7.93173E−05 
               
               
                 177 
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                 5.59102E−05 
               
               
                 178 
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                 0.000152375 
               
               
                 179 
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                 3.91975E−05 
               
               
                 180 
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                 8.48754E−05 
               
               
                 181 
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                 0.306840305 
                 3.93781E−05 
               
               
                 182 
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                 23.27621742 
                 0.000703772 
               
               
                 183 
                 5.50708E−05 
                 10.16043742 
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                 4.24145E−05 
               
               
                 184 
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                 9.79903E−05 
               
               
                 185 
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                 4.24955E−05 
               
               
                 186 
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                 3.442804843 
                 0.000549449 
               
               
                 187 
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                 0.010928806 
               
               
                 188 
                 7.19099E−05 
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                 4.57906E−05 
               
               
                 189 
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                 2.274638603 
                 0.000187553 
               
               
                 190 
                 0.000119875 
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                 1.221553255 
                  3.8427E−05 
               
               
                 191 
                 7.12456E−05 
                 11.66574302 
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                 4.19525E−05 
               
               
                 192 
                 4.19024E−05 
                 11.66574302 
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                 5.21066E−05 
               
               
                 193 
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                 3.862890569 
                 0.013431024 
               
               
                 194 
                 3.82148E−05 
                 10.16043742 
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                 3.59508E−05 
               
               
                 195 
                 0.070447558 
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                 3.068403046 
                 0.003829724 
               
               
                 196 
                 0.003421942 
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                 8.21109E−05 
               
               
                 197 
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                 4.235575283 
                 6.57536E−05 
               
               
                 198 
                 7.34401E−05 
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                 3.46408E−05 
               
               
                 199 
                 0.055210676 
                 21.22816259 
                 19.81127403 
                 0.00035693 
               
               
                 200 
                 0.001170004 
                 20.27273789 
                 1.039710441 
                 0.000150667 
               
               
                 201 
                 0.010369934 
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                 0.970314241 
                 0.001476792 
               
               
                 202 
                 9.92438E−05 
                 9.929157627 
                 0.453850068 
                 6.05564E−05 
               
               
                 203 
                 0.004408103 
                 15.02838821 
                 4.752393632 
                 0.000167575 
               
               
                 204 
                 0.000153088 
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                 6.29582E−05 
               
               
                 205 
                 0.707008218 
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                 4.235575283 
                 0.036236842 
               
               
                 206 
                 0.000782934 
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                 1.308917895 
                 7.86962E−05 
               
               
                 207 
                 0.00011326 
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                 0.433423451 
                 4.65875E−05 
               
               
                 208 
                 0.001393141 
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                 4.334234505 
                 6.15443E−05 
               
               
                 209 
                 0.001849097 
                 14.35199932 
                 5.092281523 
                 6.92015E−05 
               
               
                 210 
                 0.000271964 
                 9.055499382 
                 1.936031438 
                 4.21008E−05 
               
               
                 211 
                 0.001399652 
                 9.703142406 
                 2.672467333 
                 0.000146581 
               
               
                 212 
                 4.64163E−05 
                 9.266429059 
                 0.377496044 
                 3.67224E−05 
               
               
                 213 
                 8.55306E−05 
                 25.52186489 
                 0.243731918 
                 3.79314E−05 
               
               
                 214 
                 0.000156546 
                 5.98290911 
                 2.027273789 
                 3.50463E−05 
               
               
                 215 
                 7.72511E−05 
                 11.14069809 
                 0.433423451 
                 4.31182E−05 
               
               
                 216 
                 0.002519187 
                 11.93747308 
                 8.451083744 
                 6.70433E−05 
               
               
                 217 
                 0.001880782 
                 22.74638603 
                 5.583575658 
                 3.99758E−05 
               
               
                 218 
                 0.001112268 
                 20.74495061 
                 4.235575283 
                  3.4321E−05 
               
               
                 219 
                 0.000864187 
                 20.74495061 
                 2.672467333 
                 4.29883E−05 
               
               
                 220 
                 0.001042542 
                 23.27621742 
                 2.86360034 
                 4.24353E−05 
               
               
                 221 
                 1.36926E−05 
                 11.66574302 
                 0.078870103 
                 4.02562E−05 
               
               
                 222 
                 0.000575915 
                 14.68630033 
                 1.370605295 
                  7.6655E−05 
               
               
                 223 
                 0.000671987 
                 12.21553255 
                 2.027273789 
                  7.3438E−05 
               
               
                 224 
                 0.00481335 
                 18.06809666 
                 3.139875335 
                 0.000230241 
               
               
                 225 
                 0.012382803 
                 22.74638603 
                 4.139161818 
                 0.000357102 
               
               
                 226 
                 0.000433317 
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                 0.612226897 
                 0.000113075 
               
               
                 227 
                 0.000137795 
                 11.14069809 
                 0.475239363 
                 7.06695E−05 
               
               
                 228 
                  9.3886E−05 
                 10.63928408 
                 0.475239363 
                 4.97657E−05 
               
               
                 229 
                 0.000313999 
                 8.258713592 
                 2.494091632 
                 4.15932E−05 
               
               
                 230 
                 0.000210483 
                 7.532036315 
                 1.088710484 
                 6.96074E−05 
               
               
                 231 
                 0.000114812 
                 11.93747308 
                 0.545647796 
                  4.8006E−05 
               
               
                 232 
                 0.002931611 
                 13.39406509 
                 2.437319175 
                 0.000245254 
               
               
                 233 
                 6.24704E−05 
                 18.4889567 
                 0.249409163 
                 3.67897E−05 
               
               
                 234 
                 0.000224071 
                 25.52186489 
                 0.545647796 
                 4.29359E−05 
               
               
                 235 
                 0.000140232 
                 15.37844434 
                 0.598290911 
                 4.14082E−05 
               
               
                 236 
                 4.47327E−05 
                 11.14069809 
                 0.238183902 
                 4.59548E−05 
               
               
                 237 
                 3.15174E−05 
                 14.35199932 
                 0.172548984 
                  3.4879E−05 
               
               
                 238 
                 0.00185966 
                 24.94091632 
                 2.998557666 
                 6.71063E−05 
               
               
                 239 
                 0.000178064 
                 9.929157627 
                 1.250006885 
                 3.92323E−05 
               
               
                 240 
                 0.000131112 
                 28.6360034 
                 0.243731918 
                  5.0291E−05 
               
               
                 241 
                 0.077185871 
                 14.68630033 
                 27.98416838 
                 0.000509888 
               
               
                 242 
                 0.002460055 
                 12.21553255 
                 1.981127403 
                 0.000276036 
               
               
                 243 
                 0.000266501 
                 20.74495061 
                 0.864793477 
                 4.07598E−05 
               
               
                 244 
                 0.000595339 
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                 1.279123286 
                 7.22179E−05 
               
               
                 245 
                 7.95906E−05 
                 23.27621742 
                 0.207449506 
                 4.47518E−05 
               
               
                 246 
                 0.005975783 
                 15.37844434 
                 3.2878531 
                 0.000318703 
               
               
                 247 
                 0.002012702 
                 11.93747308 
                 6.869298762 
                 6.71892E−05 
               
               
                 248 
                 0.000702835 
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                 2.552186489 
                 5.23388E−05 
               
               
                 249 
                 0.053096317 
                 12.79123286 
                 20.74495061 
                 0.00054322 
               
               
                 250 
                 0.002184807 
                 22.74638603 
                 4.235575283 
                  6.0935E−05 
               
               
                 251 
                 0.000338308 
                 11.93747308 
                 1.573665433 
                 4.86469E−05 
               
               
                 252 
                 0.000244172 
                 11.14069809 
                 0.719303884 
                 8.35599E−05 
               
               
                 253 
                 0.017365732 
                 23.27621742 
                 3.213012427 
                 0.000631947 
               
               
                 254 
                 0.000381754 
                 13.08917895 
                 1.039710441 
                 7.64048E−05 
               
               
                 255 
                 0.000239664 
                 16.1032081 
                 0.545647796 
                 7.52696E−05 
               
               
                 256 
                 0.000165229 
                 13.70605295 
                 0.533227336 
                  6.1892E−05 
               
               
                 257 
                 0.572826298 
                 17.25489835 
                 2.274638603 
                 0.039630115 
               
               
                 258 
                 0.000443714 
                 15.02838821 
                 0.992915763 
                 7.97373E−05 
               
               
                 259 
                 0.000717834 
                 19.36031438 
                 1.936031438 
                 5.12357E−05 
               
               
                 260 
                 0.00035004 
                 11.66574302 
                 0.992915763 
                 8.13609E−05 
               
               
                 261 
                 0.003980082 
                 21.22816259 
                 4.644215946 
                 0.000109751 
               
               
                 262 
                 0.004494006 
                 13.08917895 
                 2.074495061 
                 0.000452661 
               
               
                 263 
                 0.00032321 
                 33.64437037 
                 0.306840305 
                 8.49517E−05 
               
               
                 264 
                 0.000597681 
                 15.37844434 
                 0.736058624 
                 0.000141442 
               
               
                 265 
                 0.083606849 
                 23.27621742 
                 4.435191796 
                 0.002199125 
               
               
                 266 
                 0.000248454 
                 6.869298762 
                 2.274638603 
                 4.28948E−05 
               
               
                 267 
                 0.003448544 
                 15.02838821 
                 2.798416838 
                 0.000221445 
               
               
                 268 
                 0.230806121 
                 14.35199932 
                 2.274638603 
                 0.019197571 
               
               
                 269 
                 0.005793992 
                 28.6360034 
                 4.235575283 
                 0.000132201 
               
               
                 270 
                 0.000141299 
                 15.37844434 
                 0.44351918 
                 5.65689E−05 
               
               
                 271 
                 0.007550977 
                 19.36031438 
                 2.027273789 
                 0.000522164 
               
               
                 272 
                 0.028153555 
                 10.639284082 
                 0.27273789 
                 0.000349045 
               
               
                 273 
                 8.07637E−05 
                 11.40019829 
                 0.321301243 
                 5.95633E−05 
               
               
                 274 
                 0.00237182 
                 11.66574302 
                 2.074495061 
                 0.000260883 
               
               
                 275 
                 0.062347981 
                 8.258713592 
                 2.734717094 
                 0.007492655 
               
               
                 276 
                 0.003912558 
                 21.22816259 
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                 277 
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                 278 
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                 279 
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                 280 
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                 281 
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                 282 
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                 283 
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                 284 
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                 285 
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                 286 
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                 287 
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                 288 
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                 289 
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                 290 
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                 291 
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                 292 
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                 293 
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                 294 
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                 295 
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                 296 
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                 297 
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                 298 
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                 299 
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                 300 
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                 301 
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                 302 
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                 303 
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                 304 
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                 305 
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                 306 
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                 307 
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                 308 
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                 309 
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                 310 
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                 311 
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                 312 
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                 313 
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                 314 
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                 315 
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                 316 
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                 317 
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                 318 
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                 319 
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                 320 
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                 321 
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                 322 
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                 323 
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                 324 
                 9.01036E−05 
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                 325 
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                 326 
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                 327 
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                 328 
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                 329 
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                 330 
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                 331 
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                 332 
                 7.20774E−05 
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                 333 
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                 334 
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                 335 
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                 336 
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                 337 
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                 338 
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                 339 
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                 340 
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                 341 
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                 342 
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                 343 
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                  7.3512E−05 
               
               
                 344 
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                 345 
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                 346 
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                 347 
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                 348 
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                 349 
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                 350 
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                 351 
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                 352 
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                 353 
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                 354 
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                 355 
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                 356 
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                 357 
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                 358 
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                 359 
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                 360 
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                 25.52186489 
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                 361 
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                 362 
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                 5.37078E−05 
               
               
                 363 
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                 364 
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                 365 
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                 366 
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                 367 
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                 368 
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                 369 
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                 370 
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                 371 
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                 372 
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                 373 
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                 374 
                 6.63686E−05 
                 22.22861507 
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                 3.81911E−05 
               
               
                 375 
                 7.50907E−05 
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                 3.70893E−05 
               
               
                 376 
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                 1.468630033 
                 0.000212993 
               
               
                 377 
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                 0.000651829 
               
               
                 378 
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                 0.001833678 
               
               
                 379 
                 7.85619E−05 
                 10.63928408 
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                 4.87707E−05 
               
               
                 380 
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                 3.2878531 
                 8.70516E−05 
               
               
                 381 
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                 4.08208E−05 
               
               
                 382 
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                 4.04629E−05 
               
               
                 383 
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                 384 
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                 1.537844434 
                 8.24444E−05 
               
               
                 385 
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                 5.43864E−05 
               
               
                 386 
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                 1.806809666 
                 0.000121176 
               
               
                 387 
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                 388 
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                 389 
                 9.70649E−05 
                 10.88710484 
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                 3.58878E−05 
               
               
                 390 
                 5.56156E−05 
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                 0.184889567 
                 3.84766E−05 
               
               
                 391 
                 0.000301138 
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                 7.01035E−05 
               
               
                 392 
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                 0.464421595 
                 6.96021E−05 
               
               
                 393 
                 0.086167577 
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                 42.35575283 
                 0.000428099 
               
               
                 394 
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                 4.752393632 
                  8.8303E−05 
               
               
                 395 
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                 1.140019829 
                 6.04461E−05 
               
               
                 396 
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                 2.611634549 
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                 397 
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                 5.92625E−05 
               
               
                 398 
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                 7.28681E−05 
               
               
                 399 
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                 6.59099E−05 
               
               
                 400 
                 0.001939939 
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                 3.2878531 
                 0.000185328 
               
               
                 401 
                 0.074518819 
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                 0.006598619 
               
               
                 402 
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                 6.18047E−05 
               
               
                 403 
                 0.00019918 
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                 3.91329E−05 
               
               
                 404 
                 0.0023339 
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                 3.52299807 
                 0.000125363 
               
               
                 405 
                 0.000307937 
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                 2.074495061 
                 3.99055E−05 
               
               
                 406 
                 4.35479E−05 
                 8.070722319 
                 0.344280484 
                 4.25484E−05 
               
               
                 407 
                 0.009636643 
                 10.63928408 
                 15.37844434 
                 0.000162331 
               
               
                 408 
                 0.000704922 
                 12.79123286 
                 1.64783 
                 9.09555E−05 
               
               
                 409 
                 0.082073394 
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                 3.139875335 
                 0.005307281 
               
               
                 410 
                 0.000172874 
                 11.14069809 
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                 6.78625E−05 
               
               
                 411 
                 0.002590415 
                 20.74495061 
                 5.713633843 
                 5.95983E−05 
               
               
                 412 
                 2.14123E−05 
                 10.16043742 
                 0.114001983 
                 5.01741E−05 
               
               
                 413 
                 1.79079E−05 
                 12.50006885 
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                 3.74308E−05 
               
               
                 414 
                 0.015229974 
                 16.4783 
                 7.532036315 
                 0.000340591 
               
               
                 415 
                 0.001564252 
                 5.456477959 
                 2.93030216 
                 0.000265267 
               
               
                 416 
                 7.42737E−05 
                 24.94091632 
                 0.232762174 
                 3.46397E−05 
               
               
                 417 
                 0.002017239 
                 12.50006885 
                 2.437319175 
                 0.000179922 
               
               
                 418 
                 0.001073996 
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                 3.2878531 
                 6.32416E−05 
               
               
                 419 
                 0.000103914 
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                 0.321301243 
                 3.60307E−05 
               
               
                 420 
                 0.00227239 
                 15.02838821 
                 5.210895997 
                 7.79872E−05 
               
               
                 421 
                 0.003094582 
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                 5.846721472 
                 8.01705E−05 
               
               
                 422 
                 0.026822594 
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                 24.37319175 
                 0.000263402 
               
               
                 423 
                 8.06153E−05 
                 17.25489835 
                 0.321301243 
                 3.90039E−05 
               
               
                 424 
                 6.65435E−05 
                 15.02838821 
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                 3.80554E−05 
               
               
                 425 
                 8.68244E−05 
                 12.50006885 
                 0.413916182 
                 4.61813E−05 
               
               
                 426 
                 5.02296E−05 
                 9.482271919 
                 0.377496044 
                  3.7914E−05 
               
               
                 427 
                 0.005633169 
                 13.39406509 
                 15.73665433 
                 7.26803E−05 
               
               
                 428 
                 0.053561311 
                 10.88710484 
                 2.611634549 
                 0.005001157 
               
               
                 429 
                 0.000211193 
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                 1.981127403 
                 3.98133E−05 
               
               
                 430 
                 0.128441705 
                 12.50006885 
                 15.02838821 
                 0.001895798 
               
               
                 431 
                 0.00032307 
                 9.482271919 
                 1.537844434 
                 6.04888E−05 
               
               
                 432 
                 0.010134136 
                 23.81839017 
                 16.4783 
                 6.92222E−05 
               
               
                 433 
                 7.69896E−05 
                 13.70605295 
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                 4.06321E−05 
               
               
                 434 
                 0.000135597 
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                 0.377496044 
                 4.37181E−05 
               
               
                 435 
                 0.001444653 
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                 4.644215946 
                 5.67292E−05 
               
               
                 436 
                 8.44475E−05 
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                 4.85387E−05 
               
               
                 437 
                 0.000172682 
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                 4.02947E−05 
               
               
                 438 
                 0.043302564 
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                 4.334234505 
                 0.001237471 
               
               
                 439 
                 0.00083712 
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                 1.140019829 
                 0.000100867 
               
               
                 440 
                 0.110359565 
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                 23.81839017 
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                 441 
                 0.00239921 
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                 0.000138022 
               
               
                 442 
                 5.51714E−05 
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                 4.43098E−05 
               
               
                 443 
                 2.73416E−05 
                 13.70605295 
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                 4.57706E−05 
               
               
                 444 
                 7.76194E−05 
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                 4.81237E−05 
               
               
                 445 
                 0.01841864 
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                 446 
                 0.00091319 
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                 447 
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                 448 
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                 0.000258749 
               
               
                 449 
                 0.000120989 
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                  5.2939E−05 
               
               
                 450 
                 0.000949163 
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                 2.222861507 
                 7.37358E−05 
               
               
                 451 
                 0.003519884 
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                 9.09167E−05 
               
               
                 452 
                 2.91528E−05 
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                 3.93474E−05 
               
               
                 453 
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                 454 
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                 455 
                 0.011539164 
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                 456 
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                 4.32175E−05 
               
               
                 457 
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                 27.98416838 
                 0.000328666 
               
               
                 458 
                 0.019758322 
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                 8.50784E−05 
               
               
                 459 
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                 460 
                 0.000162264 
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                 8.49288E−05 
               
               
                 461 
                 8.60808E−05 
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                 3.95986E−05 
               
               
                 462 
                 0.000100838 
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                 0.5210896 
                  5.101E−05 
               
               
                 463 
                 0.026273224 
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                 6.712934432 
                 0.000467224 
               
               
                 464 
                 0.003758225 
                 13.70605295 
                 3.52299807 
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                 465 
                 0.000251714 
                 14.02530793 
                 0.948227192 
                 5.16242E−05 
               
               
                 466 
                 0.210714099 
                 20.74495061 
                 15.37844434 
                 0.001793468 
               
               
                 467 
                 0.015124482 
                 16.86212891 
                 18.4889567 
                 0.000130494 
               
               
                 468 
                 0.001275927 
                 18.91961982 
                 2.381839017 
                  7.7579E−05 
               
               
                 469 
                 0.005514392 
                 20.74495061 
                 2.494091632 
                 0.000288274 
               
               
                 470 
                 0.156586151 
                 18.4889567 
                 6.410802613 
                 0.003538119 
               
               
                 471 
                 0.186229111 
                 14.35199932 
                 2.998557666 
                 0.011991882 
               
               
                 472 
                 0.042285785 
                 28.6360034 
                 5.210895997 
                 0.000773999 
               
               
                 473 
                 0.650258534 
                 12.21553255 
                 44.35191796 
                 0.003257705 
               
               
                 474 
                 0.144329762 
                 25.52186489 
                 5.210895997 
                 0.002904785 
               
               
                 475 
                 0.002158857 
                 16.4783 
                 0.184889567 
                 0.001965935 
               
               
                 476 
                 0.006971615 
                 20.27273789 
                 2.86360034 
                 0.000330172 
               
               
                 477 
                 0.00402333 
                 9.929157627 
                 3.774960444 
                 0.00029246 
               
               
                 478 
                 0.033812192 
                 17.25489835 
                 5.98290911 
                 0.000880456 
               
               
                 479 
                 0.00023843 
                 15.02838821 
                 0.249409163 
                 0.000173268 
               
               
                 480 
                 0.001645208 
                 19.36031438 
                 1.308917895 
                 0.000179905 
               
               
                 481 
                 0.0484191 
                 11.93747308 
                 7.532036315 
                 0.001430588 
               
               
                 482 
                 9.55314E−05 
                 18.4889567 
                 0.184889567 
                 7.59792E−05 
               
               
                 483 
                 0.041845605 
                 11.93747308 
                 9.703142406 
                 0.000984742 
               
               
                 484 
                 0.019450519 
                 19.81127403 
                 5.456477959 
                 0.000480395 
               
               
                 485 
                 0.026711018 
                 8.647934772 
                 2.798416838 
                 0.002995786 
               
               
                 486 
                 0.002357911 
                 18.4889567 
                 2.437319175 
                 0.000142909 
               
               
                 487 
                 0.421294865 
                 33.64437037 
                 25.52186489 
                 0.001344337 
               
               
                 488 
                 0.025919091 
                 16.1032081 
                 30.68403046 
                 0.000141309 
               
               
                 489 
                 0.00481277 
                 27.34717094 
                 0.433423451 
                 0.001102531 
               
               
                 490 
                 0.005726208 
                 9.266429059 
                 5.98290911 
                 0.000280416 
               
               
                 491 
                 0.016151725 
                 15.37844434 
                 3.862890569 
                 0.000738517 
               
               
                 492 
                 0.000487614 
                 7.360586237 
                 3.442804843 
                 5.22113E−05 
               
               
                 493 
                 0.061733956 
                 29.3030216 
                 6.410802613 
                 0.000885952 
               
               
                 494 
                 0.001663264 
                 19.81127403 
                 1.502838821 
                 0.000152943 
               
               
                 495 
                 0.066165089 
                 11.40019829 
                 19.81127403 
                 0.000798045 
               
               
                 496 
                 0.014395555 
                 15.37844434 
                 14.68630033 
                 0.000170788 
               
               
                 497 
                 0.009138115 
                 16.4783 
                 2.381839017 
                 0.000638927 
               
               
                 498 
                 0.105623599 
                 15.02838821 
                 7.029305285 
                 0.002777924 
               
               
                 499 
                 0.000110394 
                 28.6360034 
                 0.279841684 
                  3.7797E−05 
               
               
                 500 
                 0.044368613 
                 9.703142406 
                 24.37319175 
                 0.000512563 
               
               
                 501 
                 0.092445553 
                 13.39406509 
                 27.34717094 
                 0.000694007 
               
               
                 502 
                 0.24858678 
                 10.39710441 
                 4.235575283 
                 0.015327737 
               
               
                 503 
                 0.000134276 
                 11.40019829 
                 0.509228152 
                 6.18108E−05 
               
               
                 504 
                 0.003680974 
                 26.11634549 
                 3.862890569 
                 9.72953E−05 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Genes expressed predominately in the Retinal Ganglion Cell  
               
               
                 Layer (RGL). Genes expressed at least at 10 fold higher  
               
               
                 levels in the GCL than in other parts of the retina, as 
               
               
                 identified both by SAM and t-test, and grouped by putative function.  
               
               
                 Promoter sequences belonging to any of these genes would in  
               
               
                 drive high and preferential gene expression in GCL 
               
               
                 and may hence be utilised to drive expression of OphNDI1  
               
               
                 contemplated in this patent application. In addition, additional  
               
               
                 genes expresses in addition to OphNDI1 such as those 
               
               
                 described in Table 6 may be expressed from any of these promoters. 
               
               
                 Table adapted from Kim et al., Mol Vis 2006; 12:1640-1646 
               
             
          
           
               
                 Transcriptional regulation and RNA 
                   
               
               
                 binding molecules 
                 ECM organisation 
               
               
                   
               
               
                 EBF 
                 CTHRC1 
               
               
                 ERF5A2 
                 LAMA4 
               
               
                 ELAVL2 
                 SERPINE2 
               
               
                 ELAVL4 
                 Neuronal development 
               
               
                 FKBP1B 
                 CRTAC1 
               
               
                 KIAA1045 
                 GAP43 
               
               
                 POU4F1 
                 NRG1 
               
               
                 RBPMS 
                 NRN1 
               
               
                 RBPMS2 
                 Fatty acid metabolism 
               
               
                 TGFB1I1 
                 FABP3 
               
               
                 Cytoskeleton/Neurofilaments 
                 LSS 
               
               
                 EPPK1 
                 Signal transduction 
               
               
                 KEF5A 
                 GPR54 
               
               
                 MAP1A 
                 RGS1 
               
               
                 MICAL2 
                 RGS5 
               
               
                 NEF3 
                 RIT2 
               
               
                 NEFH 
                 Apoptosis 
               
               
                 NEFL 
                 IER3 
               
               
                 PRPH 
                 LGALS1 
               
               
                 TMSB10 
                 TNFRSF21 
               
               
                 Endocytosis/neurotransmitter 
                 Miscellaneous 
               
               
                 transport/synaptic transmission 
                   
               
               
                 ANXA2 
                 GGH 
               
               
                 AP1G1 
                 HBA2 
               
               
                 CHRNB3 
                 HHL 
               
               
                 CPLX1 
                 HLA-DPA1 
               
               
                 GNAS 
                 LMO2 
               
               
                 QPRT 
                 MT3 
               
               
                 RAB13 
                 PECAM1 
               
               
                 STMN2 
                 PPP2R2C 
               
               
                 STXBP6 
                 UCHL1 
               
               
                 SYNGR3 
                 Cell adhesion 
               
               
                 Ion/Anion transport 
                 FAT3 
               
               
                 ATP1B1 
                 FN1 
               
               
                 KCNA2 
                 GJA1 
               
               
                 KCNJ8 
                 PCDH7 
               
               
                 SCN1A 
                 SRPX 
               
               
                 SCN1B 
                 THY1 
               
               
                 SCN4B 
                   
               
               
                 SLC17A6 
                   
               
               
                 SLC4A11 
                   
               
               
                 GABRB3 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Transcripts detected at very high levels by gene array analyses  
               
               
                 of the human retinal ganglion cell layer (GCL). The genes listed  
               
               
                 here are likely to represent highly abundant 
               
               
                 transcripts of the ganglion cell layer. Promoter  
               
               
                 sequences belonging to any of these genes 
               
               
                 would in theory drive very high levels of gene  
               
               
                 expression in GCL and may hence be utilised 
               
               
                 to drive expression of OphNDI1 and the  
               
               
                 contemplated in this patent application. In addition, 
               
               
                 additional genes expresses in addition to  
               
               
                 OphNDI1 such as those described in Table 6 may be 
               
               
                 expressed from any of these promoters. 
               
               
                 Table adapted from Kim et al., Mol Vis 2006;12:1640-1646 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 TF 
                 H3F3A 
               
               
                   
                 TUBA3 
                 COX7A2 
               
               
                   
                 NEFH 
                 RTN1 
               
               
                   
                 GABARAPL3 
                 CALM2 
               
               
                   
                 TUBB 
                 MAFF 
               
               
                   
                 GLUL 
                 INA 
               
               
                   
                 UBB 
                 PGK1 
               
               
                   
                 NEFL 
                 AF1Q 
               
               
                   
                 EIF3S6IP 
                 YWHAB 
               
               
                   
                 PGAM1 
                 SUI1 
               
               
                   
                 LDHA 
                 DDAH1 
               
               
                   
                 RTN4 
                 EIF4A2 
               
               
                   
                 HINT1 
                 MAP1B 
               
               
                   
                 LDHB 
                 NDUFB8 
               
               
                   
                 PGR1 
                 K-ALPHA-1 
               
               
                   
                 EEF1A1 
                 STK35 
               
               
                   
                 PTPRO 
                 NEF3 
               
               
                   
                 SNAP25 
                 TMSB10 
               
               
                   
                 FTH1 
                 DRLM 
               
               
                   
                 EEF1D 
                 MGC14697 
               
               
                   
                 SKP1A 
                 FTL 
               
               
                   
                 BEX1 
                 CSRP2 
               
               
                   
                 HSPA8 
                 SRP14 
               
               
                   
                 PCP4 
                 CYCS 
               
               
                   
                 PARK7 
                 BNIP3 
               
               
                   
                 MAP4 
                 LAMP1 
               
               
                   
                 ACTG1 
                 WIF1 
               
               
                   
                 CDIPT 
                 MDH1 
               
               
                   
                 VAMP1 
                 NARS 
               
               
                   
                 SMT3H2 
                 OAZ1 
               
               
                   
                 EEF1G 
                 STOM 
               
               
                   
                 COX5A 
                 GNAS 
               
               
                   
                 SPARCL1 
                 NGFRAP1 
               
               
                   
                 UBC 
                 DBI 
               
               
                   
                 KARS 
                 TSC22 
               
               
                   
                 C6orf53 
                 ATP6V0E 
               
               
                   
                 VEGF 
                 FDFT1 
               
               
                   
                 COX4I1 
                 SAT 
               
               
                   
                 STMN2 
                 ATP5A1 
               
               
                   
                 NPM1 
                 MTCH1 
               
               
                   
                 APP 
                 HIG1 
               
               
                   
                 CIRBP 
                 GPX3 
               
               
                   
                 B2M 
                 CFL1 
               
               
                   
                 DP1 
                 MYL6 
               
               
                   
                 LAPTM4B 
                 SNCG 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Exemplary universal promoters, inducible/conditional promoters,  
               
               
                 enhancer elements and epigenetic elements 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Promoters 
                 Reference 
               
               
                   
                   
               
               
                   
                 chicken β-actin promoter 
                 Miyazaki et al., Gene. 1989 
               
               
                   
                   
                 July 15; 79(2):269-77. 
               
               
                   
                 SV40 promoter 
                 Byrne et al., Proc Natl Acad 
               
               
                   
                   
                 Sci USA. 1983 
               
               
                   
                   
                 February; 80(3):721-5. 
               
               
                   
                 CMV promoter 
                 Thomsen et al., Proc Natl 
               
               
                   
                   
                 Acad Sci USA. 1984 
               
               
                   
                   
                 February; 81(3):659-63. 
               
               
                   
                   
                 Schmidt et al., Mol Cell. Biol. 
               
               
                   
                   
                 August 1990 vol.10 no.8 
               
               
                   
                   
                 4406-4411. 
               
               
                   
                   
                 Furth et al., Nucl Acids Res. 
               
               
                   
                   
                 (1991) 19(22):6205-6208. 
               
               
                   
                 Ubiquitin promoter 
                 Schorpp et al., Nucl. Acids 
               
               
                   
                   
                 Res. (1996) 24 (9):1787- 
               
               
                   
                   
                 1788. 
               
               
                   
                 PGK promoter 
                 McBurney et al., Dev Dyn. 
               
               
                   
                   
                 August 1994; 200(4):278-93. 
               
               
                   
                   
               
               
                   
                 Inducible Promoters 
                 Reference 
               
               
                   
                   
               
               
                   
                 tetR 
                 Steiger et al., 2007 
               
               
                   
                   
               
               
                   
                 Enhancer Element 
                 Reference 
               
               
                   
                   
               
               
                   
                 Chicken ovalbumin upstream  
                 Eguchi et al., Biochimie 
               
               
                   
                 promoter transcription factor II 
                 89(3):278-88, 2007 
               
               
                   
                 Mouse dystrophin muscle  
                 Anderson et al., Mol. Ther. 
               
               
                   
                 promoter/enhancer 
                 14(5):724-34, 2006 
               
               
                   
                 Tobacco eIF4A-10  
                 Tian et al., J. Plant Physiol. 
               
               
                   
                 promoter elements 
                 162(12):1355-66, 2005 
               
               
                   
                 Immunoglobulin (Ig)  
                 Frezza et al., Ann. Rheum. 
               
               
                   
                 enhancer element HS1, 2A 
                 Dis. Mar. 28, 2007 
               
               
                   
                 Col9a1 enhancer element 
                 Genzer and Bridgewater 
               
               
                   
                   
                 Nucleic Acids Res. 
               
               
                   
                   
                 35(4):1178-86, 2007 
               
               
                   
                 Gata2 intronic enhancer 
                 Khandekar et al., 
               
               
                   
                   
                 Development Mar. 29, 2007 
               
               
                   
                 TH promoter enhancer 
                 Gao et al., Brain Res. 
               
               
                   
                   
                 1130(1):1-16, 2007 
               
               
                   
                 CMV enhancer 
                 InvivoGen cat# pdrive-cag 
               
               
                   
                   
                 05A13-SV 
               
               
                   
                 Woodchuck hepatitis virus  
                 Donello et al., J. Virol. 
               
               
                   
                 posttranscriptional  
                 72(6):5085-92, 1998 
               
               
                   
                 regulatory element 
                   
               
               
                   
                 Woodchuck hepatitis virus  
                 Schambach et al., Gene Ther. 
               
               
                   
                 posttranscriptional  
                 13(7):641-5, 2006 
               
               
                   
                 regulatory element 
                   
               
               
                   
                 IRBP 
                 Ying et al., Curr. Eye Res. 
               
               
                   
                   
                 17(8):777-82, 1998 
               
               
                   
                 CMV enhancer and  
                 InvivoGen cat# pdrive-cag 
               
               
                   
                 chicken β-actin promoter 
                 05A13-SV 
               
               
                   
                 CMV enhancer and chicken  
                 InvivoGen cat# pdrive-cag 
               
               
                   
                 β-actin promoter and 5&#39;UTR 
                 05A13-SV 
               
               
                   
                 CpG-island 
                 Antoniou et al., Genomics 
               
               
                   
                   
                 82:269-279, 2003 
               
               
                   
                   
               
               
                   
                 Epigenetic elements 
                 Reference 
               
               
                   
                   
               
               
                   
                 Mcp Insulators 
                 Kyrchanova et al., Mol. Cell Biol. 
               
               
                   
                   
                 27(8):3035-43, 2007 
               
               
                   
                 CpG-island region of  
                 Williams et al., BMC Biotechnol.  
               
               
                   
                 the HNRPA2B1 locus 
                 5:17, 2005 
               
               
                   
                 Chicken b-globin  
                 Kwaks and Otte 2006 Trends in 
               
               
                   
                 5&#39;hypersensitive site 4 (cHS4) 
                 Biotechnology 24:137-142 
               
               
                   
                 Ubiquitous chromatin  
                 Kwaks and Otte 2006 Trends in 
               
               
                   
                 opening elements (UCOEs) 
                 Biotechnology 24:137-142 
               
               
                   
                 Matrix associated  
                 Kwaks and Otte 2006 Trends in 
               
               
                   
                 regions (MARs) 
                 Biotechnology 24:137-142 
               
               
                   
                 Stabilising and antirepressor  
                 Kwaks and Otte 2006 Trends in 
               
               
                   
                 elements (STAR) 
                 Biotechnology 24:137-142 
               
               
                   
                 Human growth  
                 Trujillo MA et al. 2006 Mol  
               
               
                   
                 hormone gene silencer 
                 Endocrinol 20:2559 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Exemplary Vectors 
               
               
                   
               
             
             
               
                 Viral Vectors 
               
             
          
           
               
                 Delivery Method 
                 Serotype 
                 Reference 
               
               
                   
               
               
                 AAV (ssAAV  
                 All serotypes,  
                 Lebkowski et al., Mol. Cell 
               
               
                 or scAAV) 
                 including  
                 Biol. 8(10):3988-96, 1988 
               
               
                   
                 but not limited to 
                 Flannery et al., Proc. Natl. 
               
               
                   
                 1, 2, 3, 4, 5, 6, 7,  
                 Acad. Sci. U.S.A. 
               
               
                   
                 8, 9, 10, 11, 12, 
                 94(13):6916-21, 1997 
               
               
                 Lentivirus (for example  
                 VSV-G 
                 Pang et al., Mol. Vis. 12: 
               
               
                 but not exclusively  
                 Rabies-G 
                 756-67, 2006 
               
               
                 Feline-FIV, 
                 Further  
                 Takahashi Methods Mol.  
               
               
                 Equine-EIAV,  
                 serotypes** 
                 Biol. 246:439-49, 2004 
               
               
                 Bovine-BIV 
                   
                 Balaggan et al., J. Gene  
               
               
                 and Simian-SIV). 
                   
                 Med. 8(3):275-85, 2006 
               
               
                 Adenovirus 
                   
                 Bennett et al., Nat. Med. 
               
               
                   
                   
                 2(6):649-54, 1996 
               
               
                 Simian papovirius  
                   
                 Kimchi-Sarfaty et al., Hum. 
               
               
                 SV40 
                   
                 Gene Ther. 13(2):299-310, 
               
               
                   
                   
                 2002 
               
               
                 Semliki Forest Virus 
                   
                 DiCiommo et al., Invest. 
               
               
                   
                   
                 Ophthalmol. Vis. Sco. 
               
               
                   
                   
                 45(9):3320-9, 2004 
               
               
                 Sendai Virus 
                   
                 Ikeda et al., Exp. Eye Res. 
               
               
                   
                   
                 75(1):39-48, 2002 
               
               
                   
               
             
          
           
               
                 The list provided is not exhaustive; other viral vectors and derivatives,  
               
               
                 natural or synthesized could be used in the invention. 
               
               
                 Non Viral Vectors or Delivery Methods 
               
             
          
           
               
                 Delivery Method 
                 Reference 
               
               
                   
               
               
                 Cationic liposomes 
                 Sakurai et al., Gene Ther. 8(9):677-86, 2001 
               
               
                 HVJ liposomes 
                 Hangai et al., Arch. Ophthalmol. 116(3):342-8, 
               
               
                   
                 1998 
               
               
                 Polyethylenimine 
                 Liao and Yau Biotechniques 42(3):285-6, 2007 
               
               
                 DNA nanoparticles 
                 Farjo et al., PloS ONE 1:e38, 2006 
               
               
                 Dendrimers 
                 Marano et al., Gene Ther. 12(21):1544-50, 
               
               
                   
                 2005 
               
               
                 Bacterial 
                 Brown and Giaccia Cancer Res. 58(7):1408-16, 
               
               
                   
                 1998 
               
               
                 Macrophages 
                 Griffiths et al., Gene Ther. 7(3):255-62, 2000 
               
               
                 Stem cells 
                 Hall et al., Exp. Hematol. 34(4):433-42, 2006 
               
               
                 Retinal transplant 
                 Ng et al., Chem. Immunol. Allergy 92:300-16, 
               
               
                   
                 2007 
               
               
                 Marrow/Mesenchymal  
                 Kicic et al., J. Neurosci. 23(21):7742-9, 2003 
               
               
                 stromal cells 
                 Chng et al., J. Gene Med. 9(1):22-32, 2007 
               
               
                 Implant (e.g.,  
                 Montezuma et al., Invest. Ophthalmol. Vis. Sci. 
               
               
                 Poly(imide)uncoated  
                 47(8):3514-22, 2006 
               
               
                 or coated) 
                   
               
               
                 Electroporation 
                 Featherstone A. Biotechnol. Lab. 11(8):16, 
               
               
                   
                 1993 
               
               
                 Targeting peptides  
                 Trompeter et al., J. Immunol Methods. 274(1- 
               
               
                 (for example but 
                 2):245-56, 2003 
               
               
                 not exclusively Tat) 
                   
               
               
                 Lipid mediated  
                 Nagahara et al., Nat. Med. 4(12):1449-52, 1998 
               
               
                 (e.g., DOPE, PEG) 
                 Zeng et al., J. Virol. 81(5):2401-17, 2007 
               
               
                   
                 Caplen et al., Gene Ther. 2(9):603-13, 
               
               
                   
                 1995Manconi et al., Int. J. Pharm. 234(1- 
               
               
                   
                 2):237-48, 2006 
               
               
                   
                 Amrite et al., Invest. Ophthalmol. Vis. Sci. 
               
               
                   
                 47(3):1149-60, 2006 
               
               
                   
                 Chalberg et al., Invest. Ophthalmol. Vis. Sci. 
               
             
          
           
               
                   
                 46(6):2140-6, 2005 
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Exemplary neurotrophic factors, anti-apoptotic agents and antioxidants.  
               
               
                 Neurotrophic factor genes, anti-apoptotic agents or antioxidants  
               
               
                 which may be used in conjunction with the optimised NdiI therapy  
               
               
                 contemplated in this patent application. These genes may be delivered  
               
               
                 at the same time as the NdiI therapy or at a different time, using the 
               
               
                 same vector as the NdiI therapy or a different one. Neurotrophic factor,  
               
               
                 anti-apoptotic agents or antioxidants genes may be expressed from  
               
               
                 ubiquitously expressed promoters such as CMV and Ubiquitin (Table 4)  
               
               
                 or from one of the promoters described in Tables 2 and 3. 
               
             
          
           
               
                 Neurotrophic factor 
                 Reference 
               
               
                   
               
               
                 NGF 
                 Carmignoto et al., 1989 
               
               
                 b-NGF 
                 Lipps 2002 
               
               
                 NT-3 
                 Lu et al., 2011 
               
               
                 NT4 
                 Krishnamoorthy et al., 2001 
               
               
                 BDNF 
                 Krishnamoorthy et al., 2001; DiPolo et al., 
               
               
                   
                 1998; Garcia and Sharma 1998; Carmignoto 
               
               
                   
                 et al., 1989 
               
               
                 GDNF 
                 Wu et al., 2004, Frasson et al., 1999, 
               
               
                   
                 Gregory-Evans et al., 2009 
               
               
                 NTN (Neurturin) 
                 Koeberle et al 2002 
               
               
                 aFGF and bFGF 
                 Faktorovich et al. 1900; LaVail et al., 
               
               
                   
                 1991, 1992 Perry et al., 1995; McLaren and 
               
               
                   
                 Inana 1997; Akimoto et al., 1999; Uteza et 
               
               
                   
                 al., 1999; Lau et al., 2000 
               
               
                 LIF 
                 Joly et al., 2008, Rhee and Yang, 2010 
               
               
                 CNTF 
                 Sieving et al., 2006, Thanos et al., 2009, Li et 
               
               
                   
                 al., 2011 
               
               
                 Hepatocyte growth factor 
                 Tönges et al., 2011 
               
               
                 PDGF 
                 Akiyaman et al., 2006 
               
               
                 VEGF 
                 Trujillo et al., 2007 
               
               
                 PEDF 
                 Cayouette et al., 1999 
               
               
                 RdCVF 
                 Leveillard et al., 2004 
               
               
                 Chondroitinase ABC 
                 Liu 2011 
               
               
                 Erythropoietin 
                 Rex et al., 2009, Rong et al., 2011, Gong et al 
               
               
                   
                 2011, Hu et al., 2011, Sullivan et al., 2011 
               
               
                 Suberythropoietc Epo 
                 Wang et al., 2011 
               
               
                   
               
               
                 Anti-apoptotic agents 
                 Reference 
               
               
                   
               
               
                 Calpain inhibitor I 
                 McKenan et al., 2007 
               
               
                 Calpain inhibitor II 
                 McKenan et al., 2007 
               
               
                 Calpeptin 
                 McKenan et al., 2007 
               
               
                 PARP 
                   
               
               
                 Norgestrel 
                 Doonan et al., 2011 
               
               
                   
               
               
                 Antioxidant 
                 Reference 
               
               
                   
               
               
                 Vitamin C 
                 www.nei.nih.gov/amd 
               
               
                 Vitamin E 
                 www.nei.nih.gov/amd 
               
               
                 Beta-carotene 
                 www.nei.nih.gov/amd 
               
               
                 SOD2 +/− catalase 
                 Jung et al., 2007, Usui et al., 2009, Doonan  
               
               
                   
                 al., 2009 
               
               
                 Rosiglitazone 
                 Doonan et al., 2009 
               
               
                 Sestrin-1 
                 Budanov et al., 2002, 2004 
               
               
                 PPAR 
                 Aoun et al., 2003, Zhao et al., 2006 
               
               
                   
                 Tomita et al., 2005, Komeina et al., 2006, 2007 
               
               
                 Lutein 
                 Li et al., 2010 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Disease phenotypes and genotypes associated with mitochondrial disease. 
               
               
                 Clinical Phenotypes (non-LHON) Associated with mtDNA Polypeptide Gene Mutations 
               
               
                 (http://www.mitomap.org/bin/view.pl/MITOMAP/ClinicalPhenotypesPolypeptide) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                   
                 Nucleotide 
                   
               
               
                 Syndromes 
                 Locus 
                 Disease* 
                 Allele 
                 Change 
                 AA Change 
               
               
                   
               
               
                 Dystonia 
                 MTND 
                 Adult-Onset Dystonia 
                 A3796G 
                 A-G 
                 T164A 
               
               
                   
                 1 
                   
                   
                   
                   
               
               
                 Dystonia, Leigh 
                 MTND 
                 LS/Dystonia 
                 T14487C 
                 T-C 
                 M63V 
               
               
                 Syndrome 
                 6 
                   
                   
                   
                   
               
               
                 Dystonia, Leigh 
                 MTND 
                 LDYT/LS 
                 G14459A 
                 G-A 
                 A72V 
               
               
                 Syndrome 
                 6 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTND 
                 LS 
                 T10158C 
                 T-C 
                 S34P 
               
               
                   
                 3 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTND 
                 LS-like/ESOC 
                 T10191C 
                 T-C 
                 S45P 
               
               
                   
                 3 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTND 
                 LS 
                 C11777A 
                 C-A 
                 R340S 
               
               
                   
                 4 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTND 
                 LS 
                 T12706C 
                 T-C 
                 F124L 
               
               
                   
                 5 
                   
                   
                   
                   
               
               
                 Leigh syndrome 
                 MTATP 
                 LS/FBSN 
                 T9176C 
                 T-C 
                 L217P 
               
               
                   
                 6 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTATP 
                 LS 
                 T9176G 
                 T-G 
                 L217R 
               
               
                   
                 6  
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTATP 
                 LS 
                 T9185C 
                 T-C 
                 L220P 
               
               
                   
                 6 
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTATP 
                 LS 
                 T9191C 
                 T C 
                 L222P 
               
               
                   
                 6  
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTATP 
                 LS/NARP 
                 T8993C 
                 T-C 
                 L156P 
               
               
                   
                 6  
                   
                   
                   
                   
               
               
                 Neurogenic 
                 MTATP 
                 NARP 
                 T8993G 
                 T-G 
                 L156R 
               
               
                 Muscle Weakness 
                 6 
                   
                   
                   
                   
               
               
                 Ataxia and  
                   
                   
                   
                   
                   
               
               
                 Retinitis 
                   
                   
                   
                   
                   
               
               
                 Pigmentosa 
                   
                   
                   
                   
                   
               
               
                 Leigh Syndrome 
                 MTCO3 
                 LS-like 
                 C9537ins 
                 C-CC 
                 Q111frameshift 
               
               
                   
                   
                   
                 C 
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 T3308C 
                 T C 
                 M1T 
               
               
                 MELAS 
                 1 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS/LHON 
                 G3376A 
                 G-A 
                 E24K 
               
               
                 MELAS 
                 1 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 G3697A 
                 G-A 
                 G131S 
               
               
                 MELAS 
                 1 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 G3946A 
                 G-A 
                 E214K 
               
               
                 MELAS 
                 1 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 T3949C 
                 T-C 
                 Y215H 
               
               
                 MELAS 
                 1 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 A11084G 
                 A-G 
                 T109A 
               
               
                 MELAS 
                 4 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 A12770G 
                 A-G 
                 E145G 
               
               
                 MELAS 
                 5 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS/LHON/LS 
                 A13045C 
                 A-C 
                 M237L 
               
               
                 MELAS 
                 5 
                 overlap syndrome 
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS/LS 
                 A13084T 
                 A-T 
                 S250C 
               
               
                 MELAS 
                 5 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS/LS 
                 G13513A 
                 G-A 
                 D393N 
               
               
                 MELAS 
                 5 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 A13514G 
                 A-G 
                 D393G 
               
               
                 MELAS 
                 5 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTND 
                 MELAS 
                 G14453A 
                 G-A 
                 A74V 
               
               
                 MELAS 
                 6 
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTCY 
                 MELAS/PD 
                 14787del 
                 TTAA- 
                 I14frameshift 
               
               
                 MELAS 
                 B 
                   
                 4 
                 del 
                   
               
               
                 Epilepsy 
                 MTCO1 
                 Therapy-resistant 
                 C6489A 
                 C-A 
                 L196I 
               
               
                   
                   
                 Epilepsy 
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTCO1 
                 Multisystem Disorder 
                 G6930A 
                 G-A 
                 G343Ter 
               
               
                 Multisystem 
                   
                   
                   
                   
                   
               
               
                 Disorder 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTCOI 
                 Myopathy and Cortical 
                 6015del5 
                 Del 5 bp 
                 Frameshift, 42 
               
               
                 Multisystem 
                   
                 Lesions 
                   
                   
                 peptide 
               
               
                 Disorder 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy 
                 MTCO2 
                 Encephalomyopathy 
                 T7587C 
                 T-C 
                 M1T 
               
               
                 Encephalomyopathy, 
                 MTCO2 
                 Multisystem Disorder 
                 G7896A 
                 G-A 
                 W104Ter 
               
               
                 Multisystem  
                   
                   
                   
                   
                   
               
               
                 Disorder 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy,  
                 MTCO2 
                 Lactic Acidosis 
                 8042del2 
                 AT-del 
                 M153Ter 
               
               
                 Lactic 
                   
                   
                   
                   
                   
               
               
                 Acidosis 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy 
                 MTCO3 
                 Encephalomyopathy 
                 G9952A 
                 G-A 
                 W248Ter 
               
               
                 Encephalomyopathy,  
                 MTCO3 
                 MELAS/PEM/NAION 
                 T9957C 
                 T-C 
                 F251L 
               
               
                 MELAS 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy,  
                 MTATP 
                 Lactic Acidosis/ 
                 9205del2 
                 TA-del  
                 Ter227M 
               
               
                 Lactic 
                 6 
                 Seizures 
                   
                   
                   
               
               
                 Acidosis 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTCY 
                 Multisystem Disorder 
                 A15579G 
                 A-G 
                 Y278C 
               
               
                 Lactic 
                 B 
                   
                   
                   
                   
               
               
                 Acidosis 
                   
                   
                   
                   
                   
               
               
                 Encephalomyopathy, 
                 MTCY 
                 Septo-Optic Dysplasia 
                 T14849C 
                 T-C 
                 S35P 
               
               
                 Septo-Optic  
                 B 
                   
                   
                   
                   
               
               
                 Dysplasia 
                   
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G14846A 
                 G-A 
                 G34S 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 Mitochondrial 
                 MTCY 
                 MM 
                 G15059A 
                 G-A 
                 G190Ter 
               
               
                 Myopathy 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15084A 
                 G-A 
                 W113Ter 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15150A 
                 G-A 
                 W135Ter 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15168A 
                 G-A 
                 W141Ter 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 T15197C 
                 T-C 
                 S151P 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT/ 
                 G15242A 
                 G-A 
                 G166Ter 
               
               
                 Intolerance 
                 B 
                 Encephalomyopathy 
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15497A 
                 G-A 
                 G251S 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 15498del24 
                 24 bp 
                 251GDPDNYT 
               
               
                 Intolerance 
                 B 
                   
                   
                 deletion- 
                 L-del258 
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15615A 
                 G-A 
                 G290D 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTCY 
                 EXIT 
                 G15723A 
                 G-A 
                 W326Ter 
               
               
                 Intolerance 
                 B 
                   
                   
                   
                   
               
               
                 Mitochondrial 
                 MTCY 
                 MM 
                 G15762A 
                 G-A 
                 G339E 
               
               
                 Myopathy 
                 B 
                   
                   
                   
                   
               
               
                 MM, CPEO 
                 MTND 
                 CPEO 
                 T11232C 
                 T-C 
                 L140P 
               
               
                   
                 4 
                   
                   
                   
                   
               
               
                 MM, Exercise  
                 MTND 
                 EXIT 
                 G11832A 
                 G-A 
                 W358Ter 
               
               
                 Intolerance 
                 4 
                   
                   
                   
                   
               
               
                 MM, Exercise 
                 MTCO1 
                 EXIT/Myoglobinuria 
                 G5920A 
                 G-A 
                 W6Ter 
               
               
                 Intolerance 
                   
                   
                   
                   
                   
               
               
                 Mitochondrial 
                 MTCO1 
                 MM &amp; 
                 G6708A 
                 G-A 
                 G269Ter 
               
               
                 Myopathy 
                   
                 Rhabdomyolysis 
                   
                   
                   
               
               
                 Mitochondrial 
                 MTCO2 
                 MM 
                 T7671A 
                 T-A 
                 M29K 
               
               
                 Myopathy 
                   
                   
                   
                   
                   
               
               
                 MM, Exercise 
                 MTCO2 
                 EXIT/Rhabdomyolysis 
                 T7989C 
                 T-C 
                 L135P 
               
               
                 Intolerance 
                   
                   
                   
                   
                   
               
               
                 Mitochondrial 
                 MTCO3 
                 Myopathy and 
                 9487del15 
                 Del 15 bp 
                 Removed 5 aa 
               
               
                 Myopathy 
                   
                 Myoglobinuria 
                   
                   
                   
               
               
                 Hypertrophic 
                 MTCY 
                 HCM 
                 G15243A 
                 G-A 
                 G166E 
               
               
                 Cardiomyopathy 
                 B 
                   
                   
                   
                   
               
               
                 Hypertrophic 
                 MTCY 
                 HCM 
                 G15498A 
                 G-A 
                 G251D 
               
               
                 Cardiomyopathy 
                 B 
                   
                   
                   
                   
               
               
                 Deafness 
                 MTCO1 
                 DEAF 
                 A7443G 
                 A-G 
                 Ter514G 
               
               
                 Deafness 
                 MTCO1 
                 DEAF 
                 1A7445C 
                 A-C 
                 Ter514S 
               
               
                 Deafness-Sensory 
                 MTCO1 
                 SNHL/LHON 
                 G7444A 
                 G-A 
                 Ter514K 
               
               
                 Neural Hearing 
                   
                   
                   
                   
                   
               
               
                 Loss 
                   
                   
                   
                   
                   
               
               
                 Deafness-Sensory 
                 MTCO1 
                 SNHL 
                 A7445G 
                 A-G 
                 Ter514Ter 
               
               
                 Neural Hearing 
                   
                   
                   
                   
                   
               
               
                 Loss 
                   
                   
                   
                   
                   
               
               
                 Deafness-Sensory 
                 MTCO2 
                 SNHL 
                 A8108G 
                 A-G 
                 I175V 
               
               
                 Neural Hearing 
                   
                   
                   
                   
                   
               
               
                 Loss 
                   
                   
                   
                   
                   
               
               
                 Deafness-Sensory 
                 MTND 
                 SNHL 
                 C14340T 
                 C-T 
                 V112M 
               
               
                 Neural Hearing 
                 6 
                   
                   
                   
                   
               
               
                 Loss 
                   
                   
                   
                   
                   
               
               
                 Diabetes Mellitus 
                 MTND 
                 NIDDM/PEO 
                 G3316A 
                 G-A 
                 A4T 
               
               
                   
                 1 
                   
                   
                   
                   
               
               
                 Diabetes Mellitus 
                 MTND 
                 DM 
                 A12026G 
                 A-G 
                 I423V 
               
               
                   
                 4 
                   
                   
                   
                   
               
               
                 Alzheimer &amp; 
                 MTND 
                 ADPD 
                 A3397G 
                 A-G 
                 M31V 
               
               
                 Parkinson Disease 
                 1 
                   
                   
                   
                   
               
               
                 Alzheimer &amp; 
                 MTND 
                 AD 
                 G5460A 
                 G-A 
                 A331T 
               
               
                 Parkinson Disease 
                 2 
                   
                   
                   
                   
               
               
                 Alzheimer &amp; 
                 MTND 
                 AD 
                 G5460T 
                 G-T 
                 A331S 
               
               
                 Parkinson Disease 
                 2 
                   
                   
                   
                   
               
               
                 Idiopathic 
                 MTCO1 
                 SIDA 
                 T6721C 
                 T-C 
                 M273T 
               
               
                 Sideroblastic 
                   
                   
                   
                   
                   
               
               
                 Anemia 
                   
                   
                   
                   
                   
               
               
                 Idiopathic 
                 MTCO1 
                 SIDA 
                 T6742C 
                 T-C 
                 I280T 
               
               
                 Sideroblastic 
                   
                   
                   
                   
                   
               
               
                 Anemia 
               
               
                   
               
             
          
           
               
                 Abbreviations  
               
               
                 ♦Plasmy: Ho, homoplasmy; He, heteroplasmy  
               
               
                 *Disease: AD, Alzheimer&#39;s Disease; ADPD, Alzheimer&#39;s Disease and Parkinsons&#39;s Disease; CPEO, Chronic Progressive External  
               
               
                 Ophthalmoplegia; EXIT, exercise intolerance; LHON Leber Hereditary Optic Neuropathy; LS, Leigh Syndrome; MELAS,  
               
               
                 Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes; MM, mitochondrial myopathy; NAION Nonarteritic 
               
               
                 Anterior Ischemic Optic Neuropathy; NARP, Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; NIDDM, Non-Insulin 
               
               
                 Dependent Diabetes Mellitus; SIDA, sideroblastic anemia; SNHL, Sensorineural Hearing Loss. 
               
               
                 ** Status: Cfrm, considered confirmed by multiple reports in the literature; Prov, provisional isolated report(s), not yet confirmed by 
               
               
                 multiple labs; P.M., reported originally in the literature at pathogenic but now generally considered to be a polymorphic variant.  
               
               
                 Clinical Phenotypes (non-LHON) Associated with mtDNA rRNA &amp; tRNA Mutations (http://www.mitomap.org/bin/view.pl/ 
               
               
                 MITOMAP/ClinicalPhenotypesRNA) 
               
             
          
           
               
                   
                 Syndromes 
                 Locus 
                 Disease* 
                 Allele 
                 RNA 
               
               
                   
               
               
                   
                 Encephalomyopathy, 
                 MTTV 
                 LS 
                 C1624T 
                 tRNA Val 
               
               
                   
                 Leigh Syndrome 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy, 
                 MTTV 
                 Adult LS 
                 G1644T 
                 tRNA Val 
               
               
                   
                 Leigh Syndrome 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTW 
                 MILS 
                 A5537insT 
                 tRNA Trp 
               
               
                   
                 Leigh Syndrome 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 MERRF 
                 A8344G 
                 ItRNA Lys 
               
               
                   
                 MERRF 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 MERRF 
                 T8356C 
                 tRNA Lys 
               
               
                   
                 MERRF 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 MERRF 
                 G8361A 
                 tRNA Lys 
               
               
                   
                 MERRF 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 MERRF/MICM+ 
                 G8363A 
                 tRNA Lys 
               
               
                   
                 MERRF 
                   
                 DEAF/Autism 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MERRF/KSS overlap 
                 G3255A 
                 tRNA Leu 
               
               
                   
                 MERRF 
                   
                   
                   
                 ( UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTF 
                 MERRF 
                 G611A 
                 tRNA Phe 
               
               
                   
                 MERRF 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTD 
                 MEPR 
                 A7543G 
                 tRNA 
               
               
                   
                 Myoclonus and 
                   
                   
                   
                 Asp 
               
               
                   
                 Psychomotor 
                   
                   
                   
                   
               
               
                   
                 Regression 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTV 
                 AMDF 
                 G1606A 
                 tRNA Val 
               
               
                   
                 Ataxia, Myoclonus 
                   
                   
                   
                   
               
               
                   
                 and Deafness 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTH 
                 MERRF-MELAS/ 
                 G12147A 
                 tRNA His 
               
               
                   
                 MERRF 
                   
                 Cerebral edema 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 A3243G 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 G3244A 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 A3252G 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 C3256T 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS/Myopathy 
                 T3258C 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 T3271C 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 MELAS 
                 T3291C 
                 tRNA Leu 
               
               
                   
                 MELAS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTV 
                 MELAS 
                 G1642A 
                 tRNA Val 
               
               
                   
                 MELAS 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTQ 
                 MELAS/ 
                 G4332A 
                 tRNA Gln 
               
               
                   
                 MELAS 
                   
                 Encephalopathy 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTF 
                 MELAS 
                 G583A 
                 tRNA Phe 
               
               
                   
                 MELAS 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTRNR 
                 MELAS 
                 C3093G 
                 16S 
               
               
                   
                 MELAS 
                 2 
                   
                   
                 rRNA 
               
               
                   
                 Encephalomyopathy 
                 MTTL1 
                 PEM 
                 T3271delT 
                 tRNA Leu 
               
               
                   
                   
                   
                   
                   
                 (UUR) 
               
               
                   
                 Encephalomyopathy 
                 MTTI 
                 Progressive  
                 T4290C 
                 tRNA Ile 
               
               
                   
                   
                   
                 Encephalopathy 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTI 
                 (Mitochondria 
                 C4320T 
                 tRNA Ile 
               
               
                   
                   
                   
                 Encephalo- 
                   
                   
               
               
                   
                   
                   
                 cardiomyopathy 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTW 
                 Encephalomyopathy 
                 G5540A 
                 tRNA Trp 
               
               
                   
                 Encephalomyopathy  
                 MTTC 
                 Encephalopathy 
                 T5814C 
                 tRNA Cys 
               
               
                   
                 Encephalomyopathy 
                 MTTS1 
                 PEM/AMDF 
                 C7472insC 
                 tRNA Ser 
               
               
                   
                   
                   
                   
                   
                 (UCN) 
               
               
                   
                 Encephalomyopathy 
                 MTTS1 
                 PEM/MERME 
                 T7512C 
                 tRNA Ser 
               
               
                   
                   
                   
                   
                   
                 (UCN) 
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 Encephalopathy 
                 C8302T 
                 tRNA Lys 
               
               
                   
                 Encephalomyopathy 
                 MTTK 
                 Mitochondrial  
                 G8328A 
                 tRNA Lys 
               
               
                   
                   
                   
                 Encephalopathy 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTG 
                 PEM 
                 T10010C 
                 tRNA Gly 
               
               
                   
                 Encephalomyopathy 
                 MTATT 
                 Encephalomyopathy 
                 G15915A 
                 tRNA Thr 
               
               
                   
                 Encepehaolmyopathy 
                 MTRNR 
                 Rett Syndrome 
                 C2835T 
                 rRNA 
               
               
                   
                 Rett Syndrome 
                 2 
                   
                   
                 16S 
               
               
                   
                 Multisystem Disease 
                 MTTI 
                 Varied familial  
                 G4284A 
                 tRNA Ile 
               
               
                   
                   
                   
                 presentation 
                   
                   
               
               
                   
                 Encephalomyopathy 
                 MTTG 
                 GER/SIDS 
                 A10044G 
                 tRNA Gly 
               
               
                   
                 Gastrointestinal 
                   
                   
                   
                   
               
               
                   
                 Reflux and Sudden 
                   
                   
                   
                   
               
               
                   
                 Infant Death 
                   
                   
                   
                   
               
               
                   
                 Syndrome 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTF 
                 MM 
                 T582C 
                 tRNA Phe 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTF 
                 MM 
                 T618C 
                 tRNA Phe 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM 
                 G3242A 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM/CPEO 
                 A3243G 
                 TRNA 
               
               
                   
                 Myopathy 
                   
                   
                   
                 Leu(UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM 
                 A3243T 
                 tRNA 
               
               
                   
                 Myopathy 
                   
                   
                   
                 Leu(UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM/CPEO 
                 T3250C 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM 
                 A3251G 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM 
                 C3254G 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 Myopathy 
                 A3280G 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 Myopathy 
                 A3288G 
                 TRNA 
               
               
                   
                 Myopathy 
                   
                   
                   
                 Leu(UUR) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MM 
                 A3302G 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTI 
                 MM 
                 A4267G 
                 tRNA Ile 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTQ 
                 Myopathy 
                 T4370AT 
                 tRNA Gln 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTM 
                 MM 
                 T4409C 
                 tRNA 
               
               
                   
                 Myopathy 
                   
                   
                   
                 Met 
               
               
                   
                 Mitochondrial 
                 MTTM 
                 MM 
                 G4450A 
                 tRNA 
               
               
                   
                 Myopathy 
                   
                   
                   
                 Met 
               
               
                   
                 Mitochondrial 
                 MTTW 
                 MM 
                 G5521A 
                 tRNA Trp 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTS1 
                 MM 
                 T7480G 
                 tRNA Ser 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UCN) 
               
               
                   
                 Mitochondrial 
                 MTTS1 
                 MM 
                 G7497A 
                 tRNA Ser 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (UCN) 
               
               
                   
                 Mitochondrial 
                 MTTK 
                 Myopathy 
                 T8355C 
                 tRNA Lys 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTK 
                 Myopathy 
                 T8362G 
                 tRNA Lys 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTG 
                 Myopathy 
                 G10014A 
                 tRNA Gly 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL2 
                 MM 
                 A12320G 
                 tRNA Leu 
               
               
                   
                 Myopathy 
                   
                   
                   
                 (CUN) 
               
               
                   
                 Mitochondrial 
                 MTTE 
                 MM + DM 
                 T14709C  
                 tRNA Glu 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTT 
                 MM 
                 T15940delT 
                 tRNA Thr 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTP 
                 MM 
                 C15990T 
                 tRNA Pro 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTY 
                 Exercise Intolerance 
                 T5874G 
                 tRNA Tyr 
               
               
                   
                 Myopathy, Exercise 
                   
                   
                   
                   
               
               
                   
                 Intolerance 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 CPEO 
                 C3254T 
                 tRNA Leu 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTI 
                 CPEO 
                 T4274C 
                 tRNA Ile 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTI 
                 CPEO 
                 T4285C 
                 tRNA Ile 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTI 
                 CPEO/MS 
                 G4298A 
                 tRNA Ile 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTI 
                 CPEO 
                 G4309A 
                 tRNA Ile 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTA 
                 CPEO 
                 T5628C 
                 tRNA Ala 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 Asn 
               
               
                   
                 Mitochondrial 
                 MTTN 
                 CPEO/MM 
                 T5692C 
                 tRNA 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 Asn 
               
               
                   
                 Mitochondrial 
                 MTTN 
                 CPEO/MM 
                 G5698A 
                 tRNA 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 Asn 
               
               
                   
                 Mitochondrial 
                 MTTN 
                 CPEO/MM 
                 G5703G 
                 tRNA 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 Asn 
               
               
                   
                 Mitochondrial 
                 MTTK 
                 CPEO + Myoclonus 
                 G8342A 
                 tRNA Lys 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL2 
                 CPEO 
                 G12294A 
                 tRNA Leu 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 (CUN) 
               
               
                   
                 Mitochondrial 
                 MTTL2 
                 CPEO/Stroke/CM 
                 A12308G 
                 tRNA Leu 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 (CUN) 
               
               
                   
                 Mitochondrial 
                 MTTL2 
                 CPEO 
                 T12311C 
                 tRNA Leu 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 (CUN) 
               
               
                   
                 Mitochondrial 
                 MTTL2 
                 CPEO 
                 G12315A 
                 tRNA Leu 
               
               
                   
                 Myopathy, CPEO 
                   
                   
                   
                 (CUN) 
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 Ocular myopathy 
                 T3273C 
                 tRNA Leu 
               
               
                   
                 Myopathy, Ocular 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 KSS 
                 G3249A 
                 tRNA Leu 
               
               
                   
                 Myopathy, KSS 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Mitochondrial 
                 MTTY 
                 Mitochondrial  
                 A5843G 
                 tRNA Tyr 
               
               
                   
                 Myopathy 
                   
                 Cytopathy/ 
                   
                   
               
               
                   
                 Cytopathy 
                   
                 FSGS 
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTK 
                 Mitochondrial  
                 A8326G 
                 tRNA Lys 
               
               
                   
                 Myopathy 
                   
                 cytopathy 
                   
                   
               
               
                   
                 Cytopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTP 
                 Mitochondrial  
                 G15995A 
                 tRNA Pro 
               
               
                   
                 Myopathy 
                   
                 cytopathy 
                   
                   
               
               
                   
                 Cytopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTF 
                 Myoglobinuria 
                 A606G 
                 TRNA 
               
               
                   
                 Myopathy with 
                   
                   
                   
                 Phe 
               
               
                   
                 Myoglobinuria 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTW 
                 Gastrointestinal  
                 G5532A 
                 tRNA Trp 
               
               
                   
                 Myopathy, 
                   
                 Syndrome 
                   
                   
               
               
                   
                 Gastrointestinal 
                   
                   
                   
                   
               
               
                   
                 Syndrome 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTK 
                 MNGIE 
                 G8313A 
                 tRNA Lys 
               
               
                   
                 Myopathy, 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                   
                   
                   
                   
               
               
                   
                 Neurogastrointestinal 
                   
                   
                   
                   
               
               
                   
                 Encephalomyopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTG 
                 CIPO 
                 A10006G 
                 tRNA Gly 
               
               
                   
                 Myopathy with 
                   
                   
                   
                   
               
               
                   
                 Chronic Intestinal 
                   
                   
                   
                   
               
               
                   
                 Pseudoobstruction 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial  
                 MTTS1 
                 CIPO 
                 C12246G 
                 tRNA Ser 
               
               
                   
                 Myopathy with 
                   
                   
                   
                 (AGY) 
               
               
                   
                 Chronic Intestinal 
                   
                   
                   
                   
               
               
                   
                 Pseudoobstruction 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTF 
                 Tubulointerstitial  
                 A608G 
                 tRNA Phe 
               
               
                   
                 Myopathy with 
                   
                 nephritis 
                   
                   
               
               
                   
                 Renal Dysfunction 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTT 
                 LIMM 
                 A15923G 
                 tRNA Thr 
               
               
                   
                 Myopathy Lethal 
                   
                   
                   
                   
               
               
                   
                 Infantile 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                   
                   
                   
                   
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTT 
                 LIMM 
                 A15924G 
                 tRNA Thr 
               
               
                   
                 Myopathy Lethal 
                   
                   
                   
                   
               
               
                   
                 Infantile 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                   
                   
                   
                   
               
               
                   
                 Myopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MMC 
                 A3260G 
                 tRNA Leu 
               
               
                   
                 Myopathy and  
                   
                   
                   
                 (UUR) 
               
               
                   
                 cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Mitochondrial 
                 MTTL1 
                 MMC 
                 C3303T 
                 tRNA Leu 
               
               
                   
                 Myopathy and  
                   
                   
                   
                 (UUR) 
               
               
                   
                 cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Maternaly Inherited 
                 MTTI 
                 MHCM 
                 A4295G 
                 tRNA Ile 
               
               
                   
                 Hypertrophic 
                   
                   
                   
                   
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Maternally Inherited 
                 MTTI 
                 MICM 
                 A4300G 
                 tRNA Ile 
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Cardiomyopathy 
                 MTTK 
                 Cardiomyopathy 
                 A8348G 
                 tRNA Lys 
               
               
                   
                 Maternally Inherited 
                 MTTG 
                 MHCM 
                 T9997C 
                 tRNA Gly 
               
               
                   
                 Hypertrophic 
                   
                   
                   
                   
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Maternally Inherited 
                 MTTH 
                 MICM 
                 G12192A 
                 tRNA His 
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Cardiomyopathy 
                 MTTL2 
                 Dilated  
                 T12297C 
                 tRNA Leu 
               
               
                   
                   
                   
                 Cardiomyopathy 
                   
                 (CUN) 
               
               
                   
                 Fatal Infantile 
                 MTTI 
                 FICP 
                 A4269G 
                 tRNA Ile 
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Plus (MELAS) 
                   
                   
                   
                   
               
               
                   
                 Fatal Infantile 
                 MTTI 
                 FICP 
                 A4317G 
                 tRNA Ile 
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Plus (MELAS) 
                   
                   
                   
                   
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 A827G 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 T961C 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 T961delT+C(n)ins 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 T961insC 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 T1005C 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 SNHL 
                 T1095C 
                 12S 
               
               
                   
                 Sensory Neural 
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Hearing Loss 
                   
                   
                   
                   
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 A1116G 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 C1494T 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTRNR 
                 DEAF 
                 A1555G 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                 rRNA 
               
               
                   
                 Deafness 
                 MTTS1 
                 SNHL 
                 T7510C 
                 tRNA Ser 
               
               
                   
                 Sensory Neural  
                   
                   
                   
                 (UCN) 
               
               
                   
                 Hearing Loss 
                   
                   
                   
                   
               
               
                   
                 Deafness 
                 MTTS1 
                 SNHL 
                 T7511C 
                 tRNA 
               
               
                   
                 Sensory Neural  
                   
                   
                   
                 Ser(UCN) 
               
               
                   
                 Hearing Loss 
                   
                   
                   
                   
               
               
                   
                 Deafness 
                 MTTS1 
                 Deafness and Cerebellar 
                 7472insC 
                 tRNA 
               
               
                   
                 cerebellar 
                   
                 Dysfunction 
                   
                 Ser(UCN) 
               
               
                   
                 dysfunction 
                   
                   
                   
                   
               
               
                   
                 Deafness 
                 MTTH 
                 DEAF + RP 
                 G12183A 
                 tRNA His 
               
               
                   
                 Deafness Ataxia and 
                 MTTE 
                 Deafness, Mental  
                 14709G 
                 tRNA Glu 
               
               
                   
                 MR 
                   
                 Retaration, 
                   
                   
               
               
                   
                   
                   
                 Cerebellar Dysfunction 
                   
                   
               
               
                   
                 Diabetes Mellitus 
                 MTRNR 
                 DM 
                 C1310T 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                   
               
               
                   
                 Diabetes Mellitus 
                 MTRNR 
                 DM 
                 A1438G 
                 12S 
               
               
                   
                   
                 1 
                   
                   
                   
               
               
                   
                 Diabetes Mellitus &amp; 
                 MTTL1 
                 DM/DMDF 
                 A3243G 
                 tRNA Leu 
               
               
                   
                 Deafness 
                   
                   
                   
                 (UUR) 
               
               
                   
                 Diabetes Mellitus 
                 MTTL1 
                 DM 
                 T3264C 
                 tRNALeu 
               
               
                   
                   
                   
                   
                   
                 (UUR) 
               
               
                   
                 Diabetes Mellitus 
                 MTTL1 
                 DM 
                 T3271C 
                 tRNA Leu 
               
               
                   
                   
                   
                   
                   
                 (UUR) 
               
               
                   
                 Diabetes Mellitus 
                 MTTI 
                 Metabolic Syndrome &amp; 
                 T4291C 
                 tRNA Ile 
               
               
                   
                 Metabolic 
                   
                 Hypomagnesemia 
                   
                   
               
               
                   
                 Syndrome 
                   
                   
                   
                   
               
               
                   
                 Diabetes Mellitus &amp; 
                 MTTK 
                 DMDF/MERRF/HCM 
                 A8296G 
                 tRNA Lys 
               
               
                   
                 Deafness &amp; 
                   
                   
                   
                   
               
               
                   
                 Cardiomyopathy 
                   
                   
                   
                   
               
               
                   
                 Diabetes Mellitus &amp; 
                 MTTS2 
                 DMDF 
                 C12258A 
                 tRNA Ser 
               
               
                   
                 Deafness and 
                   
                   
                   
                 (AGY) 
               
               
                   
                 Retinitis Pigmentosa 
                   
                   
                   
                   
               
               
                   
                 Movement Disorder 
                 MTTV 
                 Movement Disorder 
                 T1659C 
                 tRNA Val 
               
               
                   
                 Alzheimer &amp; 
                 MTRNR 
                 ADPD 
                 G3196A 
                 rRNA 
               
               
                   
                 Parkinson Disease 
                 2 
                   
                   
                 16S 
               
               
                   
                 Alzheimer &amp; 
                 MTTQ 
                 ADPD/Hearing loss and 
                 T4336C 
                 tRNA Gln 
               
               
                   
                 Parkinson Disease 
                   
                 migraine 
                   
                   
               
               
                   
                 Deafness &amp; 
                   
                   
                   
                   
               
               
                   
                 Migraine 
                   
                   
                   
                   
               
               
                   
                 Dementia and 
                 MTTW 
                 DEMCHO 
                 G5549A 
                 tRNA Trp 
               
               
                   
                 Chorea 
               
               
                   
               
             
          
           
               
                 Abbreviations 
               
               
                 Plasmy: Ho, homoplasmy; He, heteroplasmy 
               
               
                 *Disease: AD, Alzheimer&#39;s Disease; ADPD, Alzheimer&#39;s Disease and Parkinsons&#39;s Disease; CIPO Chronic Intestinal  
               
               
                 Pseudoobstruction with myopathy and Ophthalmoplegia; CPEO, Chronic Progressive External Ophthalmoplegia; DEMCHO,  
               
               
                 Dementia and Chorea; DM, Diabetes Mellitus; DMDF Diabetes Mellitus &amp; Deafness; EXIT, exercise intolerance; FBSN Familial  
               
               
                 Bilateral Striatal Necrosis; FICP Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; HCM, Hypertrophic  
               
               
                 CardioMyopathy; LS, Leigh Syndrome; MELAS, Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes;  
               
               
                 MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM Maternally Inherited Hypertrophic Cardiomyopathy; MICM  
               
               
                 Maternally Inherited Cardiomyopathy; MM, mitochondrial myopathy; NAION Nonarteritic Anterior Ischemic Optic Neuropathy;  
               
               
                 NARP, Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; NIDDM, Non-Insulin Dependent Diabetes Mellitus; SNHL, 
               
               
                 Sensorineural Hearing Loss. 
               
               
                 **Status: Cfrm, considered confirmed by multiple reports in the literature; Prov, provisional isolated report(s), not yet confirmed by 
               
               
                 multiple labs; P.M., reported originally in the literature at pathogenic but now generally considered to be a polymorphic variant. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Disease phenotypes which are associated with  
               
               
                 mitochondrial mutations and where similar phenotypes may be caused  
               
               
                 by genomic mutations. Patients with these phenotypes, 
               
               
                 whether due to mitochondrial or genomic mutations,  
               
               
                 may benefit from OphNDI1 treatment. Possible target tissues  
               
               
                 for therapies directed to these disorders are indicated. 
               
             
          
           
               
                 Disease phenotype 
                 Possible target tissue type 
               
               
                   
               
               
                 Encephalomyopathy 
                 Brain, Muscle 
               
               
                 Cardiomyopathy 
                 Muscle 
               
               
                 Myopathy 
                 Muscle 
               
               
                 Migraine 
                 Brain 
               
               
                 Gastrointestinal Reflux and  
                 Brain 
               
               
                 Sudden Infant Death Syndrome 
                   
               
               
                 Lactic Acidosis 
                 Muscle 
               
               
                 Muscle Weakness 
                 Muscle 
               
               
                 Deafness 
                 Neurons 
               
               
                 Alzheimer 
                 Brain 
               
               
                 Dementia 
                 Brain 
               
               
                 Epilepsy 
                 Brain 
               
               
                 Septo-Optic Dysplasia 
                 Brain, Optic Nerve, Pituitary 
               
               
                 Parkinson Disease 
                 Brain 
               
               
                 Anemia 
                 Bone marrow 
               
               
                 Dystonia 
                 Brain 
               
               
                 Ataxia 
                 Brain 
               
               
                 Sensory Neural Hearing Loss 
                 Neurons in ear 
               
               
                 Chorea 
                 Brain 
               
               
                 Retinitis Pigmentosa 
                 Photoreceptor cell in retina 
               
               
                 Exercise Intolerance 
                 Muscles 
               
               
                 Diabetes 
                 Pancreas 
               
               
                 Age related macular degeneration 
                 Photoreceptor cell in retina

Technology Classification (CPC): 0