NZ724376B2 - Variants of yeast ndi1 gene, and uses thereof in the treatment of disease associated with mitochondrial dysfunction - Google Patents
Variants of yeast ndi1 gene, and uses thereof in the treatment of disease associated with mitochondrial dysfunction Download PDFInfo
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- NZ724376B2 NZ724376B2 NZ724376A NZ72437612A NZ724376B2 NZ 724376 B2 NZ724376 B2 NZ 724376B2 NZ 724376 A NZ724376 A NZ 724376A NZ 72437612 A NZ72437612 A NZ 72437612A NZ 724376 B2 NZ724376 B2 NZ 724376B2
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/05—Oxidoreductases acting on NADH or NADPH (1.6) with a quinone or similar compound as acceptor (1.6.5)
- C12Y106/05009—NADH:ubiquinone reductase (non-electrogenic) (1.6.5.9)
Abstract
Disclosed is an isolated codon optimised nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a sequence having at least 95% sequence identity with SEQ ID NO: 542, that comprises at least 1 codon that is codon optimised compared with the sequence of the wild-type yeast NDI1 gene of SEQ ID NO: 1. e of SEQ ID NO: 1.
Description
Title
Variants of yeast NDI1 gene, and uses thereof in the treatment of disease associated with
mitochondrial dysfunction.
Technical Field
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
Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder affecting
1/25,000 people, predominantly males . Loss of central vision results from the
degeneration of the retinal ganglion cell (RGC) layer and optic nerve . In over 95% of
patients the genetic pathogenesis of LHON involves mutations in genes encoding
components of the mitochondrial respiratory NADH-ubiquinone oxidoreductase
complex (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 . It is perhaps
not surprising that a tissue such as retina, with the most significant energy requirements
of any mammalian tissue , 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 . 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 (OXPHOS). Mitochondrial complex I mutations leading to respiratory
chain dysfunction are hence linked to reduced oxygen consumption; a reliable measure
of overall mitochondrial activity.
Interestingly, many LHON mutations are not fully penetrant, it seems that the
appearance of the pathological features of the disorder may be influenced by genetic and
40 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 . 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
45 consumption, the efficiency of electron transport and ATP production . 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 . Nuclear
modifier genes can influence LHON progression and severity, for example, an x-linked
modifier locus has been reported . Additionally, smoking has been suggested as one of
50 the environmental factors which can influence disease penetrance . In addition, the
male prevalence (5:1) of LHON may at last in part be influenced by oestrogens . An
interplay between the primary mutation, modifying nuclear genes, the mtDNA genetic
background and environmental factors may collaborate to determine overall risk of
visual loss for a given LHON patient.
While significant progress has been made with regard to understanding the genetic
pathogenesis of LHON, development of gene therapies for LHON has been impeded by
the need to deliver therapies to the mitochondria of RGCs. In addition, intragenic
heterogeneity has made development of therapies complex. Allotopic or nuclear
expression of mitochondrial genes is being explored as a potential therapeutic avenue
for some mitochondrial disorders including ND4-linked LHON, although modifications
14,15,16
may be required to facilitate import of expressed proteins into mitochondria . A
nuclear complementation approach using NDI1 has been considered as a potential
therapy for Parkinson disease (PD) . 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 . 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.
Statements of Invention
The invention relates to variants of the yeast NDI1 gene of SEQ ID NO: 1 which are
codon optimised to provide for improved expression in mammalian cells, and/or
modified to encode an immune optimised functional variant of NDI1 protein. Codon
optimisation involves replacing codons which are common to yeast cells and uncommon
to mammalian cells with synonomous codons which are common to mammalian cells.
These are known as “silent changes” as they do not result in an amino acid change in the
encoded protein. Codon optomisation provides for improved expression of the nucleic
acid in mammalian cells and/or conveys less immunogenicity. Immune optimisation
involves substitution of one or more amino acids (i.e. see Table 1b), for example from
one to ten amino acids, in the protein to provide a variant protein that exhibits reduced
immunogenicity in-vivo in humans compared to yeast NDI1 protein. Examples of
possible amino acid changes include conservative amino acid changes at one or more of
the following positions:
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259,
K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85,
Y151, Y482, S488,V45, L483, S80, K196, for example one or more of the following amino
acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M,
K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E,
L481I, L202M, L259V, L195I , L150M, R85K, Y151F, Y482F, S488T,V45I, L483M, S80T,
K196T.
40 In an aspect, the present invention provides an isolated codon optimised nucleic acid
sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a sequence having at
least 95% sequence identity with SEQ ID NO: 542, that, compared with the sequence of
the wild-type yeast NDI1 gene of SEQ ID NO: 1, comprises at least 50 codons which are
codon optimised for expression in mammalian cells. In an embodiment, the nucleic acid
45 sequence comprises at least 100, 200, 300 or 329 codons which are codon optimised
compared with the sequence of wild-type yeast NDI1 gene of SEQ ID NO: 1. In an
embodiment, said NDI1 protein is immune optimised. In an embodiment, the isolated
nucleic acid sequence encodes an immune optimised functional variant of the yeast
NDI1 protein of SEQ ID NO: 542 comprising at least one conservative amino acid change
50 to a residue selected from the group consisting of: L19, L150, L151, L195, and L259.
In an aspect, the present invention provides a nucleic acid construct comprising the
nucleic acid sequence of the preceding aspect and a nucleic acid sequence encoding a
mitochondrial localisation sequence.
In an aspect, the present invention provides a vector suitable for use in gene therapy
and comprising a nucleic acid sequence of the preceding aspect.
In an embodiment, the vector is an adeno-associated virus (AAV). In an embodiment, the
vector comprises a nucleic acid sequence encoding a mitochondrial localisation
sequence. In an embodiment, the vector is for intraocular delivery. In an embodiment,
the vector additionally comprises the GDNF gene. In an embodiment, the vector
additionally comprises a gene that enhances cell survival and or cell function. In an
embodiment, the gene that enhances cell survival or cell function is selected from a gene
encoding a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant,
a cytokine, or a hormone. In an embodiment, the vector comprises a promotor, wherein
the at least one nucleic acid is expressed from the promotor. In an embodiment, the
promotor is one that is preferentially or specifically expressed in retinal ganglion cells
(RGC’s) wherein expression of the nucleic acid is under the control of the promotor.
In an aspect, the present invention provides a vector suitable for use in transfecting a
mammalian retinal ganglion cell and comprising a nucleic acid according to a preceding
aspect, and a nucleic acid encoding GDNF, in which the vector is an AAV virus.
In an aspect, the present invention provides a pharmaceutical formulation suitable for
intraocular delivery and comprising the vector of a preceding aspect.
In an aspect, the present invention provides a system comprising a vector of a preceding
aspect in combination with a second vector comprising the GDNF gene.
In an aspect, the present invention provides a system comprising a vector of a preceding
aspect in combination with a second vector comprising a gene that enhances cell
survival and or cell function.
In an embodiment, the second vector comprises a gene encoding a neurotrophic factor, a
growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, or a hormone.
In an aspect, the present invention provides an isolated cell transformed with a nucleic
acid of a preceding aspect or the vector of a preceding aspect or the system of a
preceding aspect.
In an embodiment, the cell is selected from a stem cell, progenitor cell, RGC, or RGC
precursor cell.
In an aspect, the present invention provides a nucleic acid of a preceding aspect, a
construct of a preceding aspect, a vector of a preceding aspect, the formulation of a
preceding aspect, the system of a preceding aspect, or the cell of a preceding aspect, for
use as a medicament.
In an aspect, the present invention provides a nucleic acid of a preceding aspect, a
construct of a preceding aspect, a vector of a preceding aspect, the formulation of a
preceding aspect, the system of a preceding aspect, or the cell of a preceding aspect, for
use in the treatment of a disease or condition associated with mitochondrial
dysfunction.
In an embodiment, the disease or condition associated with mitochondrial dysfunction
is selected from the group consisting of a neurodegenerative disease, a muscular disease
and Leber Hereditory Optic Neuropathy (LHON).
In an aspect, the present invention provides an active agent comprising a protein
encoded by the nucleic acid of a preceding aspect, the vector of a preceding aspect, or a
nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a sequence
having at least 90% sequence identity with SEQ ID NO: 542, for use in the treatment of a
disease or condition associated with mitochondrial dysfunction.
In an embodiment, the active agent further comprises an additional agent comprising
the GDNF gene. In an embodiment, the active agent further comprises an additional
agent comprising a gene or protein or compound that enhances cell survival and/or cell
function. In an embodiment, said gene or protein or compound that enhances cell
survival and/or cell function is selected from a neurotrophic factor, a growth factor, an
anti-apoptotic agent, an antioxidant, a cytokine, or a hormone. In an embodiment, the
disease or condition associated with mitochondrial dysfunction is selected from the
group comprising a neurodegenerative disease, a muscular disease and Leber
Hereditory Optic Neuropathy (LHON).
In an aspect, the present invention provides a use of a nucleic acid of a preceding aspect,
a construct of a preceding aspect, a vector of a preceding aspect, the formulation of a
preceding aspect, the system of a preceding aspect, or the cell of a preceding aspect in
the manufacture of a medicament.
In an aspect, the present invention provides a use of a nucleic acid of a preceding aspect,
a construct of a preceding aspect, a vector of a preceding aspect, the formulation of a
preceding aspect, the system of a preceding aspect, or the cell of a preceding aspect, in
the manufacture of a medicament for treating a disease or condition associated with
mitochondrial dysfunction.
In an embodiment, the disease or condition associated with mitochondrial dysfunction
40 is selected from the group consisting of a neurodegenerative disease, a muscular disease
and Leber Hereditory Optic Neuropathy (LHON).
In an aspect, the present invention provides a use of an active agent comprising a
protein encoded by the nucleic acid of a preceding aspect, the vector of a preceding
45 aspect, or a nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or
a sequence having at least 90% sequence identity with SEQ ID NO: 542, in the
manufacture of a medicament for treating a disease or condition associated with
mitochondrial dysfunction.
50 In an embodiment, the use of the active agent further comprises an additional agent
comprising the GDNF gene. In an embodiment, the use of the active agent further
comprises an additional agent comprising a gene or protein or compound that enhances
cell survival and/or cell function. In an embodiment, said gene or protein or compound
that enhances cell survival and/or cell function is selected from a neurotrophic factor, a
growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, or a hormone. In an
embodiment, the disease or condition associated with mitochondrial dysfunction is
selected from the group comprising a neurodegenerative disease, a muscular disease
and Leber Hereditory Optic Neuropathy (LHON).
In an aspect, the invention provides an isolated nucleic acid sequence encoding the yeast
NDI1 protein of SEQ ID NO: 542 or a functional variant thereof having at least 90%
sequence identity with SEQ ID NO: 542 , wherein the nucleic acid comprises at least 50
codons which are codon optimised compared with the sequence of yeast NDI1 gene of
SEQ ID NO: 1.
Examples of codon optimised variants of yeast NDI1 gene are provided in SEQ ID NO’S:
2-62, 75-145, 165-243, 264-341, 362-441, 462-541, and 705-1004 .
In an aspect, the invention provides an isolated codon optimised nucleic acid sequence
encoding an immune optimised functional variant of the yeast NDI1 protein of
SEQ ID NO: 542 comprising at least one conservative amino acid change at a residue
selected from the group consisting of :
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259,
K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85,
Y151, Y482, S488,V45, L483, S80, K196, for example one or more of the following amino
acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M,
K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E,
L481I, L202M, L259V, L195I , L150M, R85K, Y151F, Y482F, S488T,V45I, L483M, S80T,
K196T, wherein the nucleic acid comprises at least 50 codons which are codon
optimised compared with the sequence of wild-type yeast NDI1 gene of SEQ ID NO: 1.
Examples of immune and codon optimised variants of yeast NDI1 gene are provided in
SEQ ID NO’S:75-145, 165-243, 264-341, 362-441, 462-541, 566-584, 705-824, 835-884,
895-944 and 955-1004.
In an aspect, the invention provides an isolated nucleic acid sequence encoding an
immune optimised functional variant of yeast NDI1 protein of SEQ ID NO: 542 in which
the variant comprises at least one conservative amino acid change at a residue selected
from the group consisting of :
L195, K284, K10, S143, L502, L403, A387, S86, F90, L94, K196, L19, K214, K373, L259,
K511, L159, R479, L483, I82, F90, L89, V266, K214, L481, L202, L259, L195, L150, R85,
Y151, Y482, S488,V45, L483, S80, K196, for example one or more of the following amino
acid changes:
L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M,
K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E,
L481I, L202M, L259V, L195I , L150M, R85K, Y151F, Y482F, S488T,V45I, L483M, S80T,
K196T,
In an additional aspect of the invention the NDI1 gene and encoded protein are immune
optimized employing amino acid substitution(s) at one or more key NDI1 positions as
defined by K10, L19, V45, S80, I82, R85, S86, L89, F90, L94, S143, L150, Y151, L159,
L195, K196, L202, K214, L259, V266, K284, K373, A387, L403, R479, L481, Y482, L483,
S488, L502, K511.
Examples of immune optimised variants of yeast NDI1 gene (without codon
optimisation) are provided in SEQ ID NO’S: 63-74 and 547-565 (one amino acid change),
146-164 and 585-605 (two amino acid changes), 244-263 and 606-640 (three amino
acid changes), 641-675 (four amino acid changes), 342-361 and 676-696 (five amino
40 acid changes), 697-703 (six amino acid changes), 704 (seven amino acid changes) and
442-461 (ten amino acid changes).
Typically, the nucleic acid sequence of the invention encodes a functional variant of the
yeast NDI1 protein of SEQ ID NO: 542 having at last 90% sequence identity with SEQ ID
45 NO:542. Preferably, the functional variant comprises at least 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 542.
Preferably, the nucleic acid sequence of the invention encodes a yeast NDI1 protein that
includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically, from 1-20, 1-
50 15, or ideally from 1-10, amino acids are changed. The changes are suitably conservative
changes made to one or more of the residues identifed above, for example one or more
of: L195F, K284E, K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M,
K214E, K373E, L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E,
L481I, L202M, L259V, L195I , L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T,
K196T.
Preferably, the nucleic acid sequence of the invention encodes a yeast NDI1 protein that
includes at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically, from 1-20, 1-15,
or ideally from 1-10, amino acids are changed, and the changes are suitably selected at
NDI1 positions from the group: K10, L19, V45, S80, I82, R85, S86, L89, F90, L94, S143,
L150, Y151, L159, L195, K196, L202, K214, L259, V266, K284, K373, A387, L403, R479,
L481, Y482, L483, S488, L502, K511.
Suitably, the variant protein includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18,or all of the amino acid changes selected from: L195F, K284E, K10R, S143N,
L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E, L259F, K511E,
L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M, L259V, L195I ,
L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
Ideally, the variant protein includes an amino acid change selected from: L195F, K284E,
K10R, S143N, L502M, L403I, A387S, S86K, F90H, L94M, K196E, L19M, K214E, K373E,
L259F, K511E, L159M, R479Q, L483M, I82V, F90Y, L89I, V266I, K214E, L481I, L202M,
L259V, L195I , L150M, R85K, Y151F, Y482F, S488T, V45I, L483M, S80T, K196T.
Preferably, at least 90, 100, 150, 200, 250, 300, 320, or 329 codons are codon optimised
for use in a mammal. In one embodiment, 1-100, 100-200, 200-300, or 300-329 codons
are optimised. Ideally, 329 codons are optimised (see SEQ ID NO’s 62, 134-145, 225-243,
324-341, 422-441, 522-541., 566-584 and 705-824).
In another embodiment 1-100, 100-200, 200-300, or 300-329 NDI1 codons are
optimised for use in mammals and the nucleic acid sequence encodes a yeast NDI1
protein that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes. Typically,
from 1-20, 1-15, or ideally from 1-10, amino acids are changed, and the changes are
suitably selected at NDI1 positions from the group: K10, L19, V45, S80, I82, R85, S86,
L89, F90, L94, S143, L150, Y151, L159, L195, K196, L202, K214, L259, V266, K284,
K373, A387, L403, R479, L481, Y482, L483, S488, L502, K511.
Preferably, the nucleic acid of the invention encodes a variant protein having at least
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:
542.
The invention also relates to a nucleic acid construct comprising a nucleic acid sequence
40 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).
45 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
22,23
increase longevity and or reduce immunogenicity .
50 In one aspect the invention relates to delivery of RNA encoding the protein and or
protein variants of the invention.
The invention also relates to a protein encoded by a nucleic acid construct of the
invention.
The term “nucleic acid sequence of the invention” as employed hereafter should be
understood to mean either or both of the nucleic acid sequences of the invention and the
nucleic acid constructs of the invention.
The invention also relates to a nucleic acid sequence selected from SEQ ID NO’s: 1-541
and 547-1004.
The invention also relates to a protein encoded by a nucleic acid sequence of the
invention. The protein may also include one or more mitochondrial localisation
signal(s). This may be but not limited to sequences such as
MLSKNLYSNKRLLTSTNTLVRFASTRS (SEQ ID NO: 1006) or
MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 1007).
The invention also relates to a vector suitable for use in gene therapy and comprising a
nucleic acid sequence of the invention. Suitably the vector is a viral vector, typically an
adeno-associated virus (AAV), preferably AAV virus serotype 2, although other AAV
serotypes and other types of vectors may be employed such as for example other viral
vectors, non-viral vectors, naked DNA and other vectors, examples of which are listed in
Table 5. Typically, the nucleic acid of the invention is expressed singly from the vector
(single delivery vehicle). In another embodiment, the nucleic acid of the invention is
expressed together with another gene either from the single delivery vehicle or using
two delivery 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.
The invention also relates to a kit comprising a vector of the invention in combination
with a second vector comprising a gene that enhances cell survival and or cell function
such as a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a
cytokine, a hormone or others, examples of which are described in Table 6. Ideally, the
second vector comprises a gene encoding a neurotrophic factor.
In an additional aspect additional gene sequences may be expressed in the same vector
as the nucleic acid of the invention from a component such as an internal ribosome
entry site (IRES) and or may be expressed using two or multiple promoter sequences.
40 Typically, the vector of the invention comprises a promotor wherein the nucleic acid of
the invention is expressed from the promotor. Preferably, the promotor is one that is
preferentially or specifically expressed in retinal ganglion cells (RGC’s) wherein
expression of the nucleic acid of the invention is under the control of the promotor.
Examples of such promotors are described in Table 4. In an alternative embodiment, the
45 vector of the invention comprises a promotor known to be expressed at low levels in
RGC’s.
In a further embodiment, the promotor is one that is known to be expressed in multiple
cell types, examples of which are described in Table 4.
In an additional aspect, the nucleic acid of the invention is expressed from an inducible
and/or conditional promotor.
In a further embodiment, the promotor is a tissue specific and/or cell specific promotor
targeting mammalian cells other than RGC’s such as the rhodopsin promotor which
expresses in rod photoreceptor cells. Suitably, the vector comprises tissue specific
and/or cell specific promotors combined with an inducible promotor system to control
expression of the nucleic acid.
The promotors may control expression of the nucleic acid of the invention in
combination with additional genes, as described above. Alternatively, the vector may
comprise different promotors for expressing the nucleic acid of the invention and the
other genes, for example, a gene encoding a neurotrophic agent.
The invention also relates to a method for the treatment and/or prevention of a
neurodegenerative disease, especially LHON, which method comprises a step of
delivering a nucleic acid of the invention to an individual by means of intraocular, ideally
intravitreal, delivery. In one aspect a nucleic acid of the invention is delivered to an
individual by means of systemic administration.
Preferably, the step of delivering the nucleic acid of the invention involves delivering a
vector of the invention to the individual.
The invention also relates to the use of a nucleic acid of the invention, or a protein
encoded by a nucleic acid of the invention, or a vector of the invention, as a medicament.
The invention also relates to a nucleic acid sequence of the invention, or a protein
encoded by a nucleic acid sequence of the invention, or a vector of the invention, for use
in the treatment of a disease or condition associated with mitochondrial dysfunction, for
example a neurodegenerative disease, especially Leber Hereditory Optic Neuropathy
(LHON). Typically, the treatment is symptomatic or prophylactic treatment.
The invention also relates to a method of treating a disease, for example a disease
associated with mitochondrial dysfunction, for example a neurodegenerative disease, in
an individual comprising a step of administering an active agent to the individual,
typically administering the active agent to the eye, ideally to the retinal ganglion cells,
photoreceptor cells or other eye cells, in which the active agent includes a nucleic acid
sequence of the invention, a protein encoded by the nucleic acid sequence of the
invention, or a vector of the invention. The treatment may be symptomatic or
prophylactic treatment.
Typically, the active agent is administered by intra-ocular, ideally intra-vitreal and/or
40 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
45 same time or before or after each other.
Ideally, the additional agent is a gene encoding a neurotrophic factor, examples of which
are described in Table 6. The active agent may be delivered by means of a vector, or by
means of separate vectors, or by direct delivery of the additional agent. The active agent
50 may be delivered to other parts of the body involving mitochondrial dysfunction, for
example, to the brain for the treatment of neurodegenerative diseases such as
Alzheimer’s disease, Parkinson’s disease or dementia, or to photoreceptor cells for the
treatment of Retinitis Pigmentosa or Age-related macular degeneration, or to muscle
cells to treat muscle weakness and/or degeneration.
Further, the nucleic acid sequence of the invention, its protein product, or a vector of the
invention, may be delivered to the target cell or tissue at the same time or at a different
time to the additional agent.
The invention also relates to a cell, for example a stem cell or progenitor cell, RGC or
RGC precursor cell that is transformed with a nucleic acid of the invention. Cells of the
invention may be delivered to the eye via subretinal and/or intravitreal injection to
treat cells of the eye affected by mitochondrial dysfunction such as RGC dysfunction.
Alternatively, cells of the invention may be delivered to other parts of the body involving
mitochondrial dysfunction, for example to the brain for the treatment of
neurodegenerative diseases such as Alzheimer disease, Parkinsons disease or dementia,
or to photoreceptor cells for the treatment of Retinitis Pigmentosa or Age-related
macular degeneration, or to muscle cells to treat muscle weakness and/or degeneration.
Thus, the invention also relates to a transformed cell of the invention for use as a
medicament. The invention also relates to a method of treating a disease or condition
involving mitochondrial dysfunction, typically a neurodegenerative disease, suitably
LHON, comprising a step of delivering cells of the invention to the individual.
The invention also provides a pharmaceutical formulation comprising an active agent
selected from a nucleic acid of the invention, a protein encoded by the nucleic acid of the
invention, a vector of the invention, or a cell of the invention, in combination with a
pharmaceutically acceptable carrier.
Suitably, the formulation is provided in the form of a slow release capsule adapted to
release the active agent following subretinal and or intravitreal injection, or following
delivery to or close to a target tissue type/cell type (see examples in Table 7).
In an additional embodiment encapsulated cell technology is employed for delivery of
the therapy.
In one embodiment the invention provides a transgenic organ, or a transgenic non-
human animal, comprising the nucleic acids and vectors of the invention.
In another embodiment the invention may be delivered to cells with mutations in the
nuclear genome which lead to disease phenotypes which are similar to disease
phenotypes related to mitochondrial mutations. For example the disease phenotypes
described in Table 8 may all result from nuclear mutations or mitochondrial mutations
40 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
45 invention.
In a further aspect, the invention relates to a method for the treatment or prevention of
a neurodegenerative disease, especially LHON, which method comprises a step of
delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention,
50 to an individual by means of intraocular delivery, ideally intravitreal and/or subretinal
delivery.
In a yet further aspect, the invention relates to a method for the treatment or prevention
of a neurodegenerative disease, especially LHON, which method comprises a step of
delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention,
and an agent that enhances cell survival and or cell function such as a neurotrophic
factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or
others (examples of which are described in Table 6) to an individual. Treatment may be
symptomatic or prophylactic.
In a yet further aspect, the invention relates to a method for the treatment or prevention
of a neurodegenerative disease, especially LHON, which method comprises a step of
delivering a yeast NDI1 gene, or a variant thereof such as a nucleic acid of the invention
using an AAV vector, and delivery of an agent, using the same or a separate AAV vector,
that enhances cell survival and or cell function such as a neurotrophic factor, a growth
factor, an anti-apoptotic agent, an antioxidant, a cytokine, a hormone or others
(examples of which are described in Table 6) to an individual. Treatment may be
symptomatic or prophylactic.
The term “yeast NDI1 gene” refers to the wild-type Saccharomyces cerviscae NDI1 gene
shown in SEQ ID NO: 1.
The term “variant of yeast NDI1 gene ” means a variant of yeast NDI1 gene which differs
from the wild-type gene due to at least codon optimisation, immune optimisation, or
both.
The term “conservative amino acid change” should to be understood to mean that the
amino acid being introduced is similar structurally, chemically, or functionally to that
being substituted. In particular, it refers to the substitution of an amino acid of a
particular grouping as defined by its side chain with a different amino acid from the
same grouping.
The term nucleic acid means deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and
artificial nucleic acid analogs such as peptide nucleic acid (PNA), morpholino- and
locked nucleic acid, glycol nucleic acid and threose nucleic acid. Artificial nucleic acid
analogs differ from DNA and RNA as they typically contain changes to the backbone of
the molecule. Nucleic acids incorporating chemical modification(s) to DNA and RNA to
optimise delivery and or increase stability and or increase longevity and or reduce
immunogenicity are also contemplated by the term nucleic acid. Modifications, such as
phosphorothioates, boranophosphate, 2’-Amino, 2’-Fluoro, 2’-Methoxy have been made
to nucleic acids to modulate parameters such as resistance to nuclease degradation,
binding affinity and or uptake. Exemplary nucleic acid molecules for use are
phosphoramidate, phosphothioate and methylphosphonate analogs of DNA and or RNA.
40 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-carboxymethylaminomethylthiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
45 methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethylthiouracil, beta-
D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-isopentenyladenine, uraciloxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 2’-O-methyl, 5-methylthiouracil, 2-thiouracil, 4-thiouracil,
50 5-methyluracil, uracil-5oxyacetic acid methylester, uraciloxyacetic acid (v),
methylthiouracil, 3-(3-aminoNcarboxypropyl) uracil, (acp3)w, and 2,6-
diaminopurine, 2-thiourdine, 5-methyl-cytidine amongst others.
The term “codon optimised” means that a codon that expresses a bias for yeast (i.e. is
common in yeast genes but uncommon in mammalian genes) is changed to a
synonomous codon (a codon that codes for the same amino acid) that expresses a bias
for mammals. Thus, the change in codon does not result in any amino acid change in the
encoded protein.
The term “immune optimised” as applied to a variant of yeast NDI1 gene means that the
gene variant encodes a variant NDI1 protein which elicits a reduced immune response
when expressed in a mammal compared to the wild-type yeast NDI1 gene.
The term “yeast NDI1 protein” should be understood to mean the wild-type
Saccharomyces cerviscae NDI1 protein shown in SEQ ID NO: 542. The “functional
variant” should be understood to mean a variant of SEQ ID NO: 542 which retains the
functionality of yeast NDI1 protein, for example, comparable oxygen consumption
measurements in the presence of rotenone (see methods below/Figure 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.
The term “neurodegenerative disease” should be understood to mean a disease
characterised by neuronal injury or death, or axonal degeneration, and includes diseases
such as motor neuron disease; prion disease; Huntington’s disease; Parkinson’s disease;
Parkinson’s plus; Tauopathies; Chromosome 17 dementias; Alzheimer’s disease;
Multiple sclerosis (MS); hereditary and acquired neuropathies; retinopathies and
diseases involving cerebellar degeneration.
In the context of the present invention, the term “gene therapy” refers to treatment of
40 individual which involves insertion of a gene into an individual’s cells for the purpose of
preventing or treating disease. Insertion of the gene is generally achieved using a
delivery vehicle, also known as a vector. Viral and non-viral vectors may be employed to
deliver a gene to a patients’ cells. Other types of vectors suitable for use in gene therapy
are described below.
The term “neurotrophic agent” should be understood to mean a protein that induces the
survival, development and function of neurons. Examples include nerve growth factor
(NGF) and brain-derived neurotrophic factor (BDNF). Other examples are provided
below.
Retinal ganglion cells (RGCs) are types of neurons located close to the inner surface (the
retinal ganglion layer) of the retina of the eye. They collectively image forming and non-
image forming visual information from the retina to several regions in the thalamus,
hypothalamus, and mid-brain.
It will be appreciated that the nucleci acids of the invention may include one or more
polyadenylation signals, typically located at the 3’- end of the molecule. In addition, the
nucleic acid may include a leader sequence and/or a stop codon. It will also be
appreciated that the nucleci acids of the invention may include one or more signals to
facilitate import of proteins into mitochondria.
Proteins and polypeptides (including variants and fragments thereof) of and for use in
the invention may be generated wholly or partly by chemical synthesis or by expression
from nucleic acid. The proteins and peptides of and for use in the present invention can
be readily prepared according to well-established, standard liquid or, preferably, solid-
phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company,
Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide
Synthesis, Springer Verlag, New York (1984).
Apart from the specific delivery systems embodied below, various delivery systems are
known and can be used to administer the therapeutic of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of
expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,
J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a
retroviral or other vector, etc. Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds may be administered by any
convenient route, for example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and may be administered together with other biologically active agents. Administration
can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical
compositions of the invention into the central nervous system by any suitable route,
including intraventricular and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached to a reservoir, such as
an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent. In addition, naked DNA
can be used for delivery.
In one aspect of the invention, agents such as surfactants may be included in
formulations to minimize aggregation of the therapeutic of the invention, whether viral
and/or non-viral vectors, proteins or polypeptides and/or cells.
40 In a specific embodiment, it may be desirable to administer the pharmaceutical
compositions of the invention locally to the area in need of treatment; this may be
achieved, for example, by means of an implant, said implant being of a porous, non-
porous, or gelatinous material, including membranes, such as sialastic membranes, or
fibers.
In another embodiment, the therapeutic can be delivered in a vesicle, in particular a
liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New
York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the therapeutic can be delivered in a controlled release
system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.
Ref. Biomed., Eng. 14:201 (1987); Buchwald et al., Surgery 88:75 (1980); Saudek et al.,
N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be
used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres.,
Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190
(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105
(1989)). In yet another embodiment, a controlled release system can be placed in
proximity of the therapeutic target, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-
138 (1984)). Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
The present invention also provides pharmaceutical compositions comprising a nucleic
acid of the invention and/or a protein encoded by the nucleic acid. Such compositions
comprise a therapeutically effective amount of the therapeutic, and a pharmaceutically
acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals,
and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the therapeutic is administered. Such pharmaceutical carriers can be sterile
liquids, such as water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations
and the like. The composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.
W. Martin. Such compositions will contain a therapeutically effective amount of the
therapeutic, preferably in purified form, together with a suitable amount of carrier so as
to provide the form for proper administration to the patient.
In a preferred embodiment, the composition is formulated in accordance with routine
procedures as a pharmaceutical composition adapted for intravenous administration to
human beings. Typically, compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also include a
45 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
50 dispensed with an infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an ampoule of sterile water
for injection or saline can be provided so that the ingredients may be mixed prior to
administration.
The amount of the therapeutic of the invention which will be effective in the treatment
of a particular disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques. In addition, in vivo
and/or in vitro assays may optionally be employed to help predict optimal dosage
ranges. The precise dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each patient's circumstances.
Nucleic Acid Sequences of the Invention
The sequence listing below provides a number of nucleic acid sequences according to
the invention, specifically:
SEQ ID NO: 1 – Yeast NDI1 gene – 0 amino acid changes – 0 codon changes
SEQ ID NO’S 2-21 and 825-834 – Yeast NDI1 gene - 0 amino acid changes – 100 codon
changes
SEQ ID NO’S 22-41and 885-894 – Yeast NDI1 gene - 0 amino acid changes – 200 codon
changes
SEQ ID NO’S 42-61 and 945-954– Yeast NDI1 gene - 0 amino acid changes – 300 codon
changes
SEQ ID NO 62 – Yeast NDI1 gene - 0 amino acid changes – 329 codon changes
SEQ ID NO’S 63-74 and 547-565– Yeast NDI1 gene - 1 amino acid changes – 0 codon
changes
SEQ ID NO’S 75-94 and 835-844 – Yeast NDI1 gene - 1 amino acid changes – 100 codon
changes
SEQ ID NO’S 95-114 and 895-904 – Yeast NDI1 gene - 1 amino acid changes – 200 codon
changes
SEQ ID NO’S 115-134 and 955-964 – Yeast NDI1 gene - 1 amino acid changes – 300
codon changes
SEQ ID NO’S 134-145 and 566-584 – Yeast NDI1 gene - 1 amino acid changes – 329
codon changes
SEQ ID NO’S 146-164 and 585-605 – Yeast NDI1 gene - 2 amino acid changes – 0 codon
changes
SEQ ID NO’S 165-184 and 845-854 – Yeast NDI1 gene - 2 amino acid changes – 100
codon changes
SEQ ID NO’S 185-204 and 905-914 – Yeast NDI1 gene - 2 amino acid changes – 200
codon changes
SEQ ID NO’S 205-224 and 965-974 – Yeast NDI1 gene - 2 amino acid changes – 300
40 codon changes
SEQ ID NO’S 225-243 and 705-725 – Yeast NDI1 gene - 2 amino acid changes – 329
codon changes
SEQ ID NO’S 244-263 and 606-640 - Yeast NDI1 gene - 3 amino acid changes – 0 codon
45 changes
SEQ ID NO’S 264-283 and 855-864 – Yeast NDI1 gene - 3 amino acid changes – 100
codon changes
SEQ ID NO’S 284-303 and 915-924 – Yeast NDI1 gene - 3 amino acid changes – 200
codon changes
50 SEQ ID NO’S 304-323 and 975-984 – Yeast NDI1 gene - 3 amino acid changes – 300
codon changes
SEQ ID NO’S 324-341 and 726-760 – Yeast NDI1 gene - 3 amino acid changes – 329
codon changes
SEQ ID NO’S 641-675 – Yeast NDI1 gene - 4 amino acid changes – 0 codon changes
SEQ ID NO’S 865-874 – Yeast NDI1 gene - 4 amino acid changes – 100 codon changes
SEQ ID NO’S 925-934 – Yeast NDI1 gene - 4 amino acid changes – 200 codon changes
SEQ ID NO’S 985-994 – Yeast NDI1 gene - 4 amino acid changes – 300 codon changes
SEQ ID NO’S 761-795 – Yeast NDI1 gene - 4 amino acid changes – 329 codon changes
SEQ ID NO’S 342-361 and 676-696 – Yeast NDI1 gene - 5 amino acid changes – 0 codon
changes
SEQ ID NO’S 362-381 and 875-884 – Yeast NDI1 gene - 5 amino acid changes – 100
codon changes
SEQ ID NO’S 382-401 and 935-944 – Yeast NDI1 gene - 5 amino acid changes – 200
codon changes
SEQ ID NO’S 402-421 and 995-1004 – Yeast NDI1 gene - 5 amino acid changes – 300
codon changes
SEQ ID NO’S 422-441 and 796-816 – Yeast NDI1 gene - 5 amino acid changes – 329
codon changes
SEQ ID NO’S 697-703 – Yeast NDI1 gene - 6 amino acid changes – 0 codon changes
SEQ ID NO’S 817-823 – Yeast NDI1 gene - 6 amino acid changes – 329 codon changes
SEQ ID NO 704 – Yeast NDI1 gene - 7 amino acid changes – 0 codon changesSEQ ID NO
824 – Yeast NDI1 gene - 7 amino acid changes – 329 codon changes
SEQ ID NO’S 442-461 – Yeast NDI1 gene - 10 amino acid changes – 0 codon changes
SEQ ID NO’S 462-481 – Yeast NDI1 gene - 10 amino acid changes – 100 codon changes
SEQ ID NO’S 482-501 – Yeast NDI1 gene - 10 amino acid changes – 200 codon changes
SEQ ID NO’S 502-521 – Yeast NDI1 gene - 10 amino acid changes – 300 codon changes
SEQ ID NO’S 522-541 – Yeast NDI1 gene - 10 amino acid changes – 329 codon changes
SEQ ID NO: 542 – Yeast NDI1 protein – 0 amino acid changes
Brief Description of the Figures
Figure 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
40 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.
Notably OphNDI1 may contain 0-10 amino acid substitutions to modulate and immune
45 response or 1-329 altered codons, which are expressed more frequently in mammalian
cells than the wild type codons in NDI1 (Table 1a & 1b and Sequence Listing). In
addition the CMV and ubiquitin promoters may be substituted for any of the promoters
indicated in Tables 2-4 and the GDNF gene may be substituted for any gene indicated in
Table 6. Sequences for these core construct designs are presented in Table 1a & 1b and
50 the attached Sequence Listing. Notably, different polyadenalation signals may also be
utilised in the constructs described.
Figure 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 (56KDa) 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) 5µmol 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 x 10 vp AAV-NDI1 or 3 x 10 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), bp (D). Error bars represent SD values and *: p<0.001.
Figure 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 5µmol rotenone. Oxygen consumption measurements from
HeLa cells transfected with pAAV-NDI1 (C) and pAAV-EGFP (D) in the absence of
rotenone (control).
Figure 4a. Oxygraphs for NDI1 constructs. Traces showing oxygen concentration (blue
line) and oxygen consumption (red line) in media treated with 5µmol 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.
Figure 4b. Bar charts of the data sets measuring the change in oxygen consumption from
the experiments in Figure 4a 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<0.05 (*) to <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<0.02 and V45I versus Y90Y p<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.
Figure 5. Histology of NDI1 treated retinas following rotenone insult. Adult wild type
mice were intravitreally injected into contralateral eyes with 3 x 10 vp AAV-NDI1 (A)
and 1 x 10 vp AAV-EGFP, to facilitate localisation of transduced regions of the retinas, or
3 x 10 vp AAV-EGFP (B) alone (n=4). Three weeks post-injection, 1.5 nmol of rotenone
45 was administered intravitrally to both eyes. Three weeks post-rotenone treatment eyes
were enucleated, fixed, cryosectioned (12µm) 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
50 nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bar: 20 m. 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<0.001.
Figure 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 (12µm) 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<0.01.
Scale bars: 10 m (A, B and C) and 2 m (D).
Figure 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<0.01.
Figure 8. Analysis of spatial vision in NDI treated mice following rotenone insult. Adult
wild type mice were intravitrally injected into contralateral eyes with 3 x 10 vp AAV-
NDI1 or 3 x 10 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
40 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<0.001.
Figure 9a. A representative western blot of proteins extracted from HeLa cells
45 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
50 expressed from OphNDI1-HA, a humanized Ndi1 with a HA tag. Lane 5; untransfected
HeLa cells.
Figure 9b: 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<0.005). a.u.:arbitrary unit
Figure 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.
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, -actin.
B, Levels of humanised NDI1 (hNDI1) expressed in mouse retina delivered invitreally
using AAV2/2 vectors were compared to levels of unmodified NDI1 delivered also using
AAV2/2. Levels of expression are expressed in copy number per unit of the
housekeeping gene -actin. As expression levels in Figures 5A and 5B are expressed in
copy number per unit of the housekeeping gene -actin, expression levels may be
compared directly.
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 100bp 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 87bp and humanised NDI1 amplification product is 115bp. 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
Figure 10a and 10b.
40 Figure 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 (Figure
11a) or employing the MHC-I pathway using the IEDB proteasomal cleavage/TAP
transport/MHC class I combined predictor (Figure 11b). Immunogenicity scores and
amino acid positions are presented.
Figure 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
50 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,
5µmol rotenone was added and a measurement of rotenone-insensitive respiration
taken once oxygen consumption stabilized.
Figure 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<0.05 and NDI1 p<0.01).
Detailed Description of the Invention
In the present invention delivery of NDI1 constructs (Figure 1) has been used to protect
cells in the presence of a complex I inhibitor, rotenone, (Figures 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 (Figures
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, Figure 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 . 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; Figures 5-8).
In the present Application, intravitreal injection of AAV-NDI1 provided substantial
protection against rotenone-induced insult, as assessed by a variety of assays (Figures
-8). Notably, histological analyses demonstrated significant protection of both RGCs
and the optic nerve (Figures 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 (Figure 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 (Figure 8). The results highlight the potential therapeutic value of NDI1-based
therapies for LHON when intravitreally delivered using AAV2/2.
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
40 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
45 (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
50 (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
(Figures 9a, 9b and 10). A statistically significant difference in levels of expression was
obtained between wild type and optimized NDI1 constructs (Figures 9a and 9b). The
results obtained for NDI1 protein (Figure 9a and 9b) 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 (Figure 10).
In addition both the wild type and the codon-optimised NDI1 constructs have been
immuno-optimised by introducing one or more amino acid changes to modulate the
immune response(s) (Table 1a & 1b and Sequence Listing). Amino acid modifications
were undertaken subsequent to in silico analyses for potential immunogenic sites within
NDI1 (see Figures 11a and 11b, 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 x 10 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 (Figures 2-4, 12). Significant retention of oxygen consumption was
observed in cells transfected with NDI1, codon-optimised and immuno-optimised
constructs (Figures 2-4, 12), when compared to untransfected control cells.
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 (Figure 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 (Figure 10a and 10c). 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 (Figure 12) and
functional preservation in vivo using MRI analyses of wild type mice intravitreally
40 injected with AAV-NSG vector (Figure 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
45 AAV-NSG comparisons, p<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
11
lower titre of AAV-NSG than AAV-NDI1 (5.99x10 vp/ml versus 2.5 x10 vp/ml)
Suggesting that less AAV-NSG is required to mediate an equivalent beneficial effect.
Cohorts of adult wild type mice were intravitreally injected with 3ul 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 (Figure 10). Levels of expression from
different vectors could be directly compared as expression was evaluated by absolute
copy number per unit of -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 (Figure 10).
All gene therapies which deliver non-human proteins risk activation of cytotoxic T-cell
responses following presentation of peptide fragments derived from the transgenic
protein. It is therefore important to the success of the treatment that immunogenicity of
the transgenic protein is modulated. One of the most effective ways this can be done is
by searching the sequence of the protein for fragments which are likely to strongly bind
MHC-I, increasing the likelihood that they will be presented on the cell surface and so
induce an immune reaction.
This approach is complicated somewhat by the presence of many different MHC-I alleles
in the human population, each of which may have slightly different binding affinities for
different peptides.
There are established bioinformatics methods for predicting the MHC-I binding affinity
of a particular peptide, several of which are available as downloadable tools. For our
purposes, the consensus prediction method of Nielsen et al (Protein Sci. 2003
May;12(5):1007-17) was most suitable, in addition to having excellent experimentally-
validated accuracy. These tools were adapted and supporting software generated to
enable prediction of affinity for a wide variety of MHC-I alleles. The computational tool
thus generated may be applied and modified to predict other types of immune
responses.
All potential peptide fragments that could be derived from the Ndi1 protein were
assayed by the consensus prediction method for binding affinity to all well-
characterised human MHC-I proteins.
METHODS
Vector Construction and AAV Production
Yeast NDI1 (Accession No: NM_ 001182483.1) was cloned as described . 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
40 NotI sites of pcDNA3.1- (Invitrogen, Paisley, UK). A minimal poly-adenylation signal
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, CA, USA) to create pAAV-NDI; Figure 1. pAAV-EGFP
45 was cloned as previously described .
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 347bp human Ubiquitin
promoter (nucleotides 3557-3904 of accession number D63791) and a human
Neurturin polyA consisting of nucleotides 1057- 1160 of accession number AL161995
50 was cloned down-stream of the GDNF gene. This entire ubiquitin-driven GDNF cassette,
including Neurturin polyA was cloned downstream of the CMV-driven NDI1 (including
the rabbit b-globulin polyA).
Codon optimized NDI1 sequences and/or with amino acid changes to reduce
immunogenicity profiles were synthesized by Geneart Inc. These were isolated on a XbaI
and XhoI fragment and cloned into pAAV-MCS (Agilent Technologies, La Jolla, CA, USA)
and pcDNA3.1- (Invitrogen, Paisley, UK) plasmids with a CMV immediate early promoter
and minimal polyA and verified by DNA sequencing..
Recombinant AAV2/2 viruses, AAV-ND1, AAV-NSG, pAAV-NDI1 V266I, AAV-huNDI1,
pAAV-huNDI1 182V and AAV-EGFP were prepared as described , with a modified
cesium chloride gradient as described 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.
Cell Culture
Human cervical carcinoma cells (HeLa, ATCC accession no. CCL-2) were transfected with
pAAV-NDI1 or pAAV-EGFP using Lipofectamine 2000 reagent, according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). 5 x 10 cells per well were
seeded onto 6-well plates containing 1ml Dulbecco’s modified Eagle medium
supplemented with 10% calf serum, 2mM glutamine and 1mM 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 1µg pAAV-NDI1
or 1µg pAAV-EGFP in triplicate. Cells were harvested 48hrs later and the cells from each
triplicate pooled for an individual experiment, each experiment was repeated in
triplicate.
Mitochondrial isolation and Western blot analysis
Mitochondria were isolated from HeLa cells using Anti-TOM22 microbeads
(Mitochondria isolation kit, Miltenyi Biotec GmbH, Germany). Isolated mitochondria
were washed twice in PBS and homogenised in 100µl radioimmunoprecipitation assay
(RIPA) buffer (150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS,
50mM TrisCl pH 8.0 and 1 protease inhibitor cocktail tablet/10mls (Roche, Mannheim,
Germany)). The homogenate was centrifuged at 10,000g for 20min 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, CA, USA). The PVDF membrane was blocked with 5% non-fat milk in tris
buffered saline (TBS, 0.05M Tris, 150mM NaCl, pH 7.5) and 0.05% (vol/vol) Tween 20
for 1hr at room temperature. Rabbit polyclonal antibodies to NDI1 (1:500, Cambridge
Research Biochemicals, Cleveland, UK) and VDAC1 (1:1000, Abcam, Cambridge, UK)
40 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 Missouri, USA) for 2hr at room
temperature, exposed to Super-Signal chemiluminescent substrate and enhancer (Pierce
Biotechnology, Rochford, IL, USA) and signal detected using X-ray film (Kodak,
45 Rochester, NY, USA). All Western blots were repeated three times.
Respiratory analysis
Respiratory measurements were performed in DMEM at 37 C on an Oxygraph-2k
(OROBOROS® INSTRUMENTS GmbH, Innsbruck, Austria) according to the
50 manufacturer’s instructions. Briefly, each chamber was calibrated with 2mls DMEM and
stirred (200 rpm) for 1hr to saturate the media with oxygen. Parallel experiments were
run in the two chambers of the Oxygraph-2k using 1 x 10 pAAV-NDI1 or 1 x 10 pAAV-
EGFP transfected HeLa cells. Following the addition of cells to the oxygen saturated
media the chamber size was reduced to 2ml to remove air. Continuous readings were
taken to establish the fully oxygenated baseline. 2ul 5mM rotenone (5µM in 100%
ethanol) was added to 1 x 10 pAAV-NDI1 or 1 x 10 pAAV-EGFP transfected HeLa cells
prior to transfer to the requisite chambers and continuous post-rotenone readings
taken. Continuous readings were taken both with and without rotenone until oxygen
consumption stabilised. Readings were taken from three independent transfections for
each construct.
Animals and Intravitreal Injections
Wild type 129 S2/SvHsd (Harlan UK Ltd, Oxfordshire, UK) mice were maintained under
specific pathogen free (spf) housing conditions. Intravitreal injections were carried out
in strict compliance with the European Communities Regulations 2002 and 2005
(Cruelty to Animals Act) and the Association for Research in Vision and Ophthalmology
(ARVO) statement for the use of animals. Briefly, adult mice were anaesthetised and
pupils dilated as described . Using topical anaesthesia (Amethocaine), a small puncture
was made in the sclera. A 34-gauge blunt-ended microneedle attached to a 10 l
Hamilton syringe was inserted through the puncture, and 0.6µl 2.5mM rotenone
(1.5nmol) in dimethyl sulfoxide (DMSO, vehicle), 0.6µl DMSO alone or 3µl 1 x 10 vp/ml
AAV2/2 was slowly, over a two minute period, administered into the vitreous. Following
intravitreal injection, an anesthetic reversing agent (100mg/10g body weight;
Atipamezole Hydrochloride) was delivered by intraperitoneal injection. Body
temperature was maintained using a homeothermic heating device. All animal studies
have been approved by the authors’ Institutional Review Board.
RNA extraction and PCR analysis
Adult wild type mice (n=6) were intravitrally injected with 3x10 vp AAV-NDI1 while
fellow eyes received 3x10 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, CA, 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’ -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,
40 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/ l. These standard curves were included in 96-well plates that also
included RNA samples to be analysed. Hence expression levels from all constructs,
45 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 actin.
50 Histology
Eyes and optic nerves were fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at
room 4°C washed three times with PBS and cryoprotected using a sucrose gradient
(10%, 20%, 30%). 10mm sections were cut on a cryostat (HM 500 Microm, Leica, Solms,
Germany) at -20°C. Nuclei were counterstained with 4’,6-diamidinophenylindole
(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
150µm apart in the central retina (600µm 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 5mm from the optic
nerve head from 3 animals per group (n=3). Three measurements per nerve were made
approximately 150µm 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 2hr at
room temperature. Washed specimens were post-fixed in buffered 2% osmium
tetroxide, dehydrated and embedded in araldite. Ultrathin cross-sections were cut on a
vibratome (Leica VT 1000 S), analysed using a Tecnai 12 BioTwin transmission electron
microscope (FEI, Eindhoven, Holland) and imaged with a SIS MegaView III surface
channel charge-coupled device (SCCD) camera (Olympus Soft Imaging Solutions,
Münster, Germany). The total number of membrane debris particles in the images was
counted in 5 cross sections per optic nerve from 3 animals per group (n=3).
Magnetic Resonance Imaging
Optic nerve integrity in experimental and control mice was assessed by Manganese
(Mn ) 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 ions to enter excitable cells through
voltage-gated calcium channels, thus analysis of Mn 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 2µl of 20mg/ml
manganese chloride solution. For image acquisition, mice were maintained under
sedation with ketamine (375µg/10g body weight) and placed on an MRI-compatible
cradle which maintains the animal’s body temperature at 37°C (respiration and
temperature were monitored for the duration of experiment). The cradle was positioned
within the MRI scanner and an initial rapid pilot image acquired to ensure accurate
40 positioning of the mouse. Oblique coronal T -weighted 2D images were acquired using
FLASH sequence (TR/TE: 150/2.5ms; Matrix: 128x128; Field of View: 20x20mm ; Flip
Angle 50º; number of averages: 40, the pixel resolution was 0.156mm/pixel). In the
oblique coronal orientation (36º), 20 slices, each measuring 0.35mm in thickness with
0.45mm inter slice gap, were recorded for an acquisition time of 9 min 36 sec. MRI scans
45 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/).
50 Optokinetics
Optokinetic response (OKR) spatial frequency thresholds were measured blind by two
independent researchers using a virtual optokinetic system (VOS, OptoMotry,
CerebralMechanics, Lethbridge, AB, Canada). OptoMotry measures the threshold of the
mouse’s optokinetic tracking response to moving gratings. Briefly, a virtual-reality
chamber is created with four 17 inch computer monitors facing into a square and the
unrestrained mouse was placed on a platform in the centre. A video camera, situated
above the animal, provided real-time video feedback. The experimenter centred the
virtual drum on the mouse’s head and judged whether the mouse made slow tracking
movements with its head and neck. The spatial frequency threshold, the point at which
the mouse no longer tracked, was obtained by incrementally increasing the spatial
frequency of the grating at 100% contrast. A staircase procedure was used in which the
step size was halved after each reversal, and terminated when the step size became
smaller than the hardware resolution (~0.003c/d, 0.2% contrast). One staircase was
presented for each direction of rotation to measure each eye separately, with the two
staircases being interspersed.
Statistical analysis
Data sets of treated and untreated samples were pooled, averaged and standard
deviation (SD) values calculated. Statistical significance of differences between data sets
was determined by either Student's two-tailed t-test or ANOVA used with Tukey's
multiple comparison post hoc test. In addition, the Kruskall-Wallis one-way analysis of
variance was applied to the MRI data set and Mann Whitney U-tests were undertaken on
all other data sets to establish that statistical significance was maintained using
nonparametric statistical models. Analysis was performed using Prism v. 5.0c
(GraphPad Software, La Jolla, CA, USA); differences with p<0.05 were considered
statistically significant
Predictions of immunogenic codons
All potential peptide fragments that could be derived from the Ndi1 protein were
assayed by the consensus prediction method for binding affinity to all well-
characterised human MHC-I proteins. All epitopes displaying a high affinity for MHC-I
(defined as a predicted IC50 < 500nM) 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
40 original fragment. The sequence with the highest sequence similarity to the original
fragment (as defined by the BLOSUM62 matrix) was selected as the optimal substitution
for that position.
In addition to the analyses described above using information regarding MHC-1 alone,
45 immunogenicity estimation and reduction in Ndi1 was achieved via in silico modelling of
antigen presentation via the MHC-I pathway using the IEDB proteasomal cleavage/TAP
transport/MHC class I combined predictor.
As fragments of 9 amino acids in length are the most commonly presented fragments by
50 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 was generated which is proportional
to the amount of that fragment that would be displayed on the cell surface by a given
MHC-I allele, taking into account proteasomal degradation, transport and binding by
MHC-I.
An overall immunogenicity factor for the 9-mer peptide was then calculated
F = G N
p p,i i
as where 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, represents the mean amount of that fragment that would be
displayed on the surface of a cell for all MHC-I alleles, weighted by how frequently each
allele occurs in the human population.
Each amino acid position A in the Ndi1 peptide was then assigned an immunogenicity
score defined as the sum of the immunogenicity factors for all 9-mer peptides
containing that amino acid. All positions whose immunogenicity score was less than
one-fifth of the highest score were not considered further, as mutations at these
positions would not be able to significantly affect the overall immunogenicity of the
protein.
For each of the remaining positions, a BLOSUM matrix (Henikoff and Henikoff, 1992)
was used to identify potential mutations that would not be overly disruptive to the
structure or function of Ndi1. A BLOSUM matrix is calculated by aligning 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.
x, y
For two amino acids x and y, the BLOSUM score is defined as the log-likelihood of
the amino acid x replacing y or vice-versa in a given position in homologous peptides. As
B =B
x, y y,x
a direct consequence of this definition, for all x and y (in other words, all
BLOSUM matrices are symmetric).
A high BLOSUM score for an amino-acid pair indicates that mutations changing one of
those amino acids to the other are more likely to be observed in 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 ( ),
indicating the likelihood that that amino acid will remain constant between homologous
proteins.
For all possible mutations at a given position, ∆B was defined as the change in the
BLOSUM score for that mutation. More formally, given an initial amino acid x and a
∆B= B − B
x,x x,y
candidate replacement amino acid y, . All mutations for which ∆B was
40 greater than 4 were considered too disruptive to protein function and not analysed
further.
For all remaining candidate mutations, immunogenicity factors F and scores S were
recalculated for the post-mutation peptide using the IEDB predictor. The reduction in
45 immunogenicity ∆S was then determined, defined as the difference between the score S
for that position in the original peptide versus the new score S after mutation.
All possible mutations were then ranked by the metric . High values
of represent mutations which are likely to cause a large reduction in
immunogenicity with a relatively small predicted impact on protein function. Outputs
with predicted amino acids and scores are provided in Table X.
The invention is not limited to the embodiments hereinbefore described which may be
varied in construction and detail without departing from the spirit of the invention.
References
1. Yu-Wai-Man P, Griffiths PG, Chinnery PF. Mitrochondrial optic neuropathies – disease
mechanisms and therapeutic strategies. Prog Retin Eye Res. 30:81-114 (2011).
2. Chalmers RM and Schapira AH. Clinical, biochemical and molecular genetic features of
Leber’s hereditary optic neuropathy. Biochim Biophys Acta. 1410(2):147-158 (1999).
3. Mackey DA, Oostra RJ, Rosenberg T, Nikoskelainen E, Bronte-Stewart J, Poulton J,
Harding AE, Govan G, Bolhuis PA, 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 SG,Lin H,Godley BF,Boulton ME. Mitrochondrial DNA damage and its potential
role in retinal degeneration. Prog Retin Eye Res. 27(6):596-607 (2008).
. Ames A 3 . CNS energy metabolism as related to function. Brain Res Brain Res Rev.
34:42-68 (2000).
6. Yen MY, Wang AG, Wei YH. Leber’s hereditary optic neuropathy: a multifactorial
disease. Prog Retin Eye Res. 25(4):381-396 (2006).
7. Porter RK, Joyce OJ, Farmer MK, Heneghan R, Tipton KF, Andrews JF, McBennett SM,
Lund MD, Jensen CH, Melia HP. Indirect measurement of mitochondrial proton leak and
its application. Int J Obes Relat Metab Disord. 23(Suppl 6):S12-18 (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’s hereditary optic
neuropathy with the 14484 mutation. J Neuroophthalmol. 17(2):103-107(1997).
9. Marcuello A, Martínez-Redondo D, Dahmani Y, Casajús JA, Ruiz-Pesini E, Montoya J,
López-Pérez MJ, 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 ML, Huoponen K, Savontaus ML, Nikoskelainen E,
Sadun AA, Salomao SR, Belfort R Jr, Griffiths P, Man PY, de Coo RF, Horvath R, Zeviani M,
Smeets HJ, Torroni A, Chinnery PF. Clinical expression of Leber hereditary optic
neuropathy is affected by the mitrochondrial DNA-haplogroup background. Am J Hum
40 Genet. 81:228-233 (2007).
11. Shankar SP, Fingert JH, Carelli V, Valentino ML, King TM, Daiger SP, Salomao SR,
Berezovsky A, Belfort R Jr, Braun TA, Sheffield VC, Sadun AA, Stone EM. Evidence for a
novel x-linked modifier locus for leber hereditary optic neuropathy. Ophthalmic Genet.
45 29(1):17-24 (2008).
12. Tsao K, Aitken PA, Johns DR. 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 FN, Caparrotta
50 L, Martinuzzi A, Ragazzi E, Ghelli A, Sadun AA, d'Amati G, Carelli V. Oestrogens
ameliorate mitochondrial dysfunction in Leber’s hereditary optic neuropathy. Brain.
134(Pt 1):220-234 (2011).
14. Qi X, Sun L, Lewin AS, Hauswirth WW, 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 JA,
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 RD, Guy J. Leber's Hereditary Optic Neuropathy-Gene Therapy: From
Benchtop to Bedside. J Ophthalmol. 2011: 179412 [Epub ahead of print] (2011).
17. Marella M, Seo BB, Yagi T, Matsuno-Yagi A. Parkinson's disease and mitochondrial
complex I: a perspective on the Ndi1 therapy. J Bioenerg Biomembr. 41(6):493-497
(2009).
18. Marella M, Seo BB, Thomas BB, Matsuno-Yagi A, Yagi T. Successful amelioration of
mitochondrial optic neuropathy using the yeast NDI1 gene in a rat animal model. PLoS
One. 5(7):e11472 (2010).
19. Palfi A, Millington-Ward S, Chadderton N, O'Reilly M, Goldmann T, Humphries MM, Li
T, Wolfrum U, Humphries P, Kenna PF, Farrar GJ. Adeno-associated virus-mediated
rhodopsin replacement provides therapeutic benefit in mice with a targeted disruption
of the rhodopsin gene. Hum Gene Ther. 2010 Mar;21(3):311-23.
. Ayuso E, Mingozzi F, Montane J, Leon X, Anguela XM, Haurigot V, Edmonson SA,
Africa L, Zhou S, High KA, Bosch F, Wright JF High AAV vector purity results in serotype-
and tissue-independent enhancement of transduction efficiency. Gene Ther. 2010
Apr;17(4):503-10.
21. Rohr UP, Wulf MA, 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 Oct;106(1):81-8.
22.Kormann MS, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-Jonat S,
Huppmann M, Mays LE, 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 Feb;29(2):154-7. doi:
.1038/nbt.1733. Epub 2011 Jan 9.
40 23.Chaput JC, 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
Table 1a: Nucleic acid and amino acid sequences of the Invention.
Gene Amino Acid Substitution Nucleic Acid Sequence
Yeast NDI1 FYLWRILYL SEQ ID NO: 1
Yeast NDI1 codon optimised FYLWRILYL SEQ ID NO: 62
Yeast NDI1 + 1amino acid FYLWRILYL → SEQ ID NO: 63
change FYLWRILYM
Yeast NDI1 codon optimized FYLWRILYL → SEQ ID NO: 134
+ 1 amino acid change FYLWRILYM
Yeast NDI1 FYLWRILYL → SEQ ID NO: 146
+ 2 amino acid change FYLWRILYM
FLKEIPNSL →
FFKEIPNSL
Yeast NDI1 codon optimized FYLWRILYL → SEQ ID NO: 225
+ 2 amino acid change FYLWRILYM
FLKEIPNSL →
FFKEIPNSL
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.
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
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
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
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
0.000170764 11.40019829 0.656012939 6.24617E-05
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329 0.004167344 15.37844434 0.884937105 0.000832679
330 0.000131618 11.14069809 0.558357566 5.63909E-05
331 4.56581E-05 9.482271919 0.255218649 5.12428E-05
332 7.20774E-05 12.50006885 0.352299807 4.45916E-05
333 0.034758616 10.63928408 2.734717094 0.003243739
334 0.001279068 16.1032081 1.339406509 0.000164095
335 0.0067709 15.37844434 10.16043742 0.000117995
336 0.000241151 9.266429059 1.806809666 3.87283E-05
337 0.000881043 11.93747308 2.027273789 9.85572E-05
338 0.001402766 22.74638603 2.222861507 7.59423E-05
339 0.000155937 17.25489835 0.584672147 4.12205E-05
340 0.000102855 26.72467333 0.286360034 3.63759E-05
341 0.029541969 16.4783 6.122268966 0.000795475
342 0.010547896 9.055499382 32.13012427 9.92001E-05
343 0.00104485 12.50006885 3.139875335 7.3512E-05
344 0.003614546 8.84937105 2.274638603 0.000497764
345 0.001856153 15.73665433 2.86360034 0.000112033
346 0.000199744 8.451083744 1.61032081 3.99152E-05
347 3.08138E-05 8.647934772 0.255218649 3.76999E-05
348 0.000222023 9.703142406 1.250006885 5.02243E-05
349 8.82657E-05 11.66574302 0.453850068 4.50325E-05
350 2.57735E-05 10.88710484 0.172548984 3.69963E-05
351 4.03044E-05 13.39406509 0.19811274 4.13067E-05
352 0.004566689 9.482271919 3.605059237 0.000362474
353 0.001055758 13.08917895 5.210895997 4.20808E-05
354 3.05164E-05 10.88710484 0.2172263 3.53289E-05
355 0.002585554 27.34717094 2.998557666 8.70808E-05
356 0.000139406 12.21553255 0.702930528 4.36022E-05
357 0.003081322 22.22861507 2.327621742 0.000163677
358 0.000205076 14.68630033 0.558357566 6.72774E-05
359 0.000146209 33.64437037 0.32878531 3.61733E-05
360 0.012689615 12.21553255 25.52186489 0.000112607
361 0.004583891 16.4783 4.752393632 0.000158805
362 0.000214841 7.360586237 1.468630033 5.37078E-05
363 0.007450718 18.91961982 2.494091632 0.000428744
364 0.106193293 10.88710484 3.364437037 0.008050725
365 6.36614E-05 12.50006885 0.368903185 3.78588E-05
366 3.36424E-05 15.02838821 0.157366543 3.92244E-05
367 6.81265E-05 10.63928408 0.453850068 3.83103E-05
368 6.02667E-05 10.16043742 0.344280484 4.64704E-05
369 0.015366884 10.88710484 2.93030216 0.001306252
370 0.041789394 19.36031438 25.52186489 0.000230784
371 0.000222478 15.73665433 0.864793477 4.34986E-05
372 0.005150502 17.25489835 2.494091632 0.000326126
373 8.00509E-05 9.482271919 0.360505924 6.48627E-05
374 6.63686E-05 22.22861507 0.212281626 3.81911E-05
375 7.50907E-05 12.21553255 0.44351918 3.70893E-05
376 0.001539591 13.39406509 1.468630033 0.000212993
377 0.130153448 18.4889567 29.3030216 0.000651829
378 0.012114767 18.06809666 0.992915763 0.001833678
379 7.85619E-05 10.63928408 0.413916182 4.87707E-05
380 0.001795076 16.86212891 3.2878531 8.70516E-05
381 0.000130478 8.451083744 1.039710441 4.08208E-05
382 0.00011804 7.029305285 1.114069809 4.04629E-05
383 0.001199255 11.14069809 0.864793477 0.000337446
384 0.00076988 16.4783 1.537844434 8.24444E-05
385 0.000328129 11.66574302 1.402530793 5.43864E-05
386 0.001878249 23.27621742 1.806809666 0.000121176
387 0.000949237 22.74638603 0.948227192 0.000119569
388 0.000322352 11.93747308 0.686929876 0.000108257
389 9.70649E-05 10.88710484 0.686929876 3.58878E-05
390 5.56156E-05 21.22816259 0.184889567 3.84766E-05
391 0.000301138 14.02530793 0.825871359 7.01035E-05
392 0.00023197 19.36031438 0.464421595 6.96021E-05
393 0.086167577 13.08917895 42.35575283 0.000428099
394 0.00320585 20.74495061 4.752393632 8.8303E-05
395 0.000356753 14.02530793 1.140019829 6.04461E-05
396 0.003535662 11.66574302 2.611634549 0.000315439
397 0.00014032 16.4783 0.386289057 5.92625E-05
398 0.004866363 12.50006885 14.68630033 7.28681E-05
399 0.000158366 23.81839017 0.273471709 6.59099E-05
400 0.001939939 8.647934772 3.2878531 0.000185328
401 0.074518819 16.1032081 1.936031438 0.006598619
402 0.000368539 16.4783 0.970314241 6.18047E-05
403 0.00019918 8.070722319 1.725489835 3.91329E-05
404 0.0023339 14.35199932 3.52299807 0.000125363
405 0.000307937 10.16043742 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 13.39406509 3.139875335 0.005307281
410 0.000172874 11.14069809 0.612226897 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 0.103971044 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 14.02530793 3.2878531 6.32416E-05
419 0.000103914 24.37319175 0.321301243 3.60307E-05
420 0.00227239 15.02838821 5.210895997 7.79872E-05
421 0.003094582 17.65681657 5.846721472 8.01705E-05
422 0.026822594 11.14069809 24.37319175 0.000263402
423 8.06153E-05 17.25489835 0.321301243 3.90039E-05
424 6.65435E-05 15.02838821 0.313987533 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 7.360586237 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 0.377496044 4.06321E-05
434 0.000135597 22.22861507 0.377496044 4.37181E-05
435 0.001444653 14.68630033 4.644215946 5.67292E-05
436 8.44475E-05 14.02530793 0.336443704 4.85387E-05
437 0.000172682 15.02838821 0.788701025 4.02947E-05
438 0.043302564 22.22861507 4.334234505 0.001237471
439 0.00083712 19.81127403 1.140019829 0.000100867
440 0.110359565 17.25489835 23.81839017 0.000713841
441 0.00239921 15.02838821 3.139875335 0.000138022
442 5.51714E-05 11.40019829 0.299855767 4.43098E-05
443 2.73416E-05 13.70605295 0.119374731 4.57706E-05
444 7.76194E-05 13.39406509 0.32878531 4.81237E-05
445 0.01841864 18.06809666 8.451083744 0.000326235
446 0.00091319 13.70605295 1.435199932 0.00012658
447 0.005087454 18.06809666 4.538500684 0.000169647
448 0.001545552 16.86212891 0.970314241 0.000258749
449 0.000120989 10.39710441 0.584672147 5.2939E-05
450 0.000949163 15.73665433 2.222861507 7.37358E-05
451 0.003519884 15.37844434 6.869298762 9.09167E-05
452 2.91528E-05 10.63928408 0.184889567 3.93474E-05
453 0.000259943 12.50006885 0.497636704 0.000114874
454 0.069613956 9.929157627 4.044942993 0.004814647
455 0.011539164 13.39406509 8.84937105 0.000266971
456 0.000245616 15.37844434 0.992915763 4.32175E-05
457 0.064510667 18.91961982 27.98416838 0.000328666
458 0.019758322 16.1032081 38.62890569 8.50784E-05
459 0.000992578 10.39710441 0.926642906 0.000279497
460 0.000162264 13.70605295 0.377496044 8.49288E-05
461 8.60808E-05 14.68630033 0.395286885 3.95986E-05
462 0.000100838 10.16043742 0.5210896 5.101E-05
463 0.026273224 22.74638603 6.712934432 0.000467224
464 0.003758225 13.70605295 3.52299807 0.000209698
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
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 ECM organisation
binding molecules
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
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
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
Table 4 Exemplary universal promoters, inducible/conditional promoters, enhancer elements
and epigenetic elements
Promoters Reference
chicken β-actin promoter Miyazaki et al., Gene. 1989
Jul 15;79(2):269-77.
SV40 promoter Byrne et al., Proc Natl Acad
Sci USA. 1983
Feb;80(3):721-5.
CMV promoter Thomsen et al., Proc Natl
Acad Sci USA. 1984
Feb;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.
1994 Aug;200(4):278-93.
Inducible Promoters Reference
tetR Steiger et al., 2007
Enhancer Element Reference
Chicken ovalbumin upstream promoter transcription factor Eguchi et al., Biochimie
II 89(3):278-88, 2007
Enhancer Element Reference
Mouse dystrophin muscle promoter/enhancer Anderson et al., Mol. Ther.
14(5):724-34, 2006
Tobacco eIF4A-10 promoter elements Tian et al., J. Plant Physiol.
162(12):1355-66, 2005
Immunoglobulin (Ig) enhancer element HS1,2A Frezza et al., Ann. Rheum.
Dis. March 28, 2007
Col9a1 enhancer element Genzer and Bridgewater
Nucleic Acids Res.
(4):1178-86, 2007
Gata2 intronic enhancer Khandekar et al.,
Development March 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 posttranscriptional regulatory Donello et al., J. Virol.
element 72(6):5085-92, 1998
Woodchuck hepatitis virus posttranscriptional regulatory Schambach et al., Gene Ther.
element 13(7):641-5, 2006
IRBP Ying et al., Curr. Eye Res.
17(8):777-82, 1998
CMV enhancer and chicken β-actin promoter InvivoGen cat# pdrive-cag
05A13-SV
CMV enhancer and chicken β-actin promoter and 5’UTR InvivoGen cat# pdrive-cag
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 the
Chicken b-globin 5’hypersensitive site 4 Kwaks and Otte 2006 Trends in
(cHS4) Biotechnology 24: 137-142
Ubiquitous chromatin opening elements Kwaks and Otte 2006 Trends in
(UCOEs) Biotechnology 24: 137-142
Matrix associated regions (MARs) Kwaks and Otte 2006 Trends in
Biotechnology 24: 137-142
Stabilising and antirepressor elements Kwaks and Otte 2006 Trends in
(STAR) Biotechnology 24: 137-142
Human growth hormone gene silencer Trujillo MA et al. 2006 Mol Endocrinol 20:
2559
Table 5. Exemplary Vectors
Viral Vectors
Delivery Method Serotype Reference
AAV (ssAAV or scAAV) All serotypes, including but Lebkowski et al., Mol. Cell
not limited to Biol. 8(10) :3988-96, 1988
1,2,3,4,5,6,7,8,9,10,11,12,
Flannery et al., Proc. Natl.
Acad. Sci. U.S.A.
94(13) :6916-21, 1997
Lentivirus (for example but VSV-G Pang et al., Mol. Vis. 12 :756-
not exclusively Feline – FIV, 67, 2006
Rabies-G
Equine – EIAV, Bovine – BIV
Takahashi Methods Mol. Biol.
and Simian – SIV). Further serotypes**
246 :439-49, 2004
Balaggan et al., J. Gene Med.
8(3) :275-85, 2006
Adenovirus Bennett et al., Nat. Med.
2(6) :649-54, 1996
Simian papovirius SV40 Kimchi-Sarfaty et al., Hum.
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
Non Viral Vectors or Delivery Methods
Delivery Method Reference
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 stromal cells Kicic et al., J. Neurosci. 23(21) :7742-9, 2003
Chng et al., J. Gene Med. 9(1) :22-32, 2007
Implant (e.g., Poly(imide)uncoated or Montezuma et al., Invest. Ophthalmol. Vis. Sci.
coated) 47(8) :3514-22, 2006
Electroporation Featherstone A. Biotechnol. Lab. 11(8) :16,
1993
Targeting peptides (for example but Trompeter et al., J. Immunol Methods. 274(1-
not exclusively Tat) 2) :245-56, 2003
Lipid mediated (e.g., DOPE, PEG) Nagahara et al., Nat. Med. 4(12) :1449-52, 1998
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
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 et
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
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)
Nucleoti
Syndromes Locus Disease* Allele de AA Change
Change
MTND
Dystonia Adult-Onset Dystonia A3796G A-G T164A
Dystonia,Leigh MTND
LS/Dystonia T14487C T-C M63V
Syndrome 6
Dystonia,Leigh MTND
LDYT/LS G14459A G-A A72V
Syndrome 6
MTND
Leigh Syndrome LS T10158C T-C S34P
MTND
Leigh Syndrome LS-like/ESOC T10191C T-C S45P
MTND
Leigh Syndrome LS C11777A C-A R340S
MTND
Leigh Syndrome LS T12706C T-C F124L
MTATP
Leigh syndrome LS/FBSN T9176C T-C L217P
MTATP
Leigh Syndrome LS T9176G T-G L217R
MTATP
Leigh Syndrome LS T9185C T-C L220P
MTATP
Leigh Syndrome LS T9191C T-C L222P
MTATP
Leigh Syndrome LS/NARP T8993C T-C L156P
Neurogenic
Muscle Weakness
MTATP
Ataxia and NARP T8993G T-G L156R
Retinitis
Pigmentosa
C9537ins
Leigh Syndrome MTCO3 LS-like C-CC Q111frameshift
Encephalomyopat MTND
MELAS T3308C T-C M1T
hy, MELAS 1
Encephalomyopat MTND
MELAS/LHON G3376A G-A E24K
hy, MELAS 1
Encephalomyopat MTND
MELAS G3697A G-A G131S
hy, MELAS 1
Encephalomyopat MTND
MELAS G3946A G-A E214K
hy, MELAS 1
Encephalomyopat MTND
MELAS T3949C T-C Y215H
hy, MELAS 1
Encephalomyopat MTND
MELAS A11084G A-G T109A
hy, MELAS 4
Encephalomyopat MTND
MELAS A12770G A-G E145G
hy, MELAS 5
Encephalomyopat MTND MELAS/LHON/ LS
A13045C A-C M237L
hy, MELAS 5 overlap syndrome
Encephalomyopat MTND
MELAS/LS A13084T A-T S250C
hy, MELAS 5
Encephalomyopat MTND
MELAS/LS G13513A G-A D393N
hy, MELAS 5
Encephalomyopat MTND
MELAS A13514G A-G D393G
hy, MELAS 5
Encephalomyopat MTND
MELAS G14453A G-A A74V
hy, MELAS 6
Encephalomyopat MTCY 14787del TTAA-
MELAS/PD I14frameshift
hy, MELAS B 4 del
Therapy-resistant
Epilepsy MTCO1 C6489A C-A L196I
Epilepsy
Encephalomyopat
hy, Multisystem MTCO1 Multisystem Disorder G6930A G-A G343Ter
Disorder
Encephalomyopat
Myopathy and Cortical Frameshift, 42
hy, Multisystem MTCOI 6015del5 Del 5 bp
Lesions peptide
Disorder
Encephalomyopat
MTCO2 Encephalomyopathy T7587C T-C M1T
Encephalomyopat
hy, Multisystem MTCO2 Multisystem Disorder G7896A G-A W104Ter
Disorder
Encephalomyopat
hy, Lactic MTCO2 Lactic Acidosis 8042del2 AT-del M153Ter
Acidosis
Encephalomyopat
MTCO3 Encephalomyopathy G9952A G-A W248Ter
Encephalomyopat
MTCO3 MELAS/PEM/ NAION T9957C T-C F251L
hy, MELAS
Encephalomyopat
MTATP Lactic Acidosis/
hy, Lactic 9205del2 TA-del Ter227M
6 Seizures
Acidosis
Encephalomyopat
MTCY
hy, Lactic Multisystem Disorder A15579G A-G Y278C
Acidosis
Encephalomyopat
MTCY
hy, Septo-Optic Septo-Optic Dysplasia T14849C T-C S35P
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/Encephalomyopa
G15242A G-A G166Ter
Intolerance B thy
MM, Exercise MTCY
EXIT G15497A G-A G251S
Intolerance B
MM, Exercise MTCY 15498del 24 bp 251GDPDNYT
EXIT
Intolerance B 24 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
MTND
MM, CPEO CPEO T11232C T-C L140P
MM, Exercise MTND
EXIT G11832A G-A W358Ter
Intolerance 4
MM, Exercise
MTCO1 EXIT/Myoglobinuria G5920A G-A W6Ter
Intolerance
Mitochondrial MM &
MTCO1 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 Myopathy and 9487del1
MTCO3 Del 15 bp Removed 5 aa
Myopathy Myoglobinuria 5
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 A7445C A-C Ter514S
Deafness-Sensory
Neural Hearing MTCO1 SNHL/LHON G7444A G-A Ter514K
Loss
Deafness-Sensory
Neural Hearing MTCO1 SNHL A7445G A-G Ter514Ter
Loss
Deafness-Sensory
Neural Hearing MTCO2 SNHL A8108G A-G I175V
Loss
Deafness-Sensory
MTND
Neural Hearing SNHL C14340T C-T V112M
Loss
MTND
Diabetes Mellitus NIDDM/PEO G3316A G- A A4T
MTND
Diabetes Mellitus DM A12026G A-G I423V
Alzheimer & MTND
ADPD A3397G A-G M31V
Parkinson Disease 1
Alzheimer & MTND
AD G5460A G-A A331T
Parkinson Disease 2
Alzheimer & MTND
AD G5460T G-T A331S
Parkinson Disease 2
Idiopathic
Sideroblastic MTCO1 SIDA T6721C T-C M273T
Anemia
Idiopathic
Sideroblastic MTCO1 SIDA T6742C T-C I280T
Anemia
Abbreviations
Plasmy: Ho, homoplasmy; He, heteroplasmy
* Disease: AD, Alzheimer's Disease; ADPD, Alzheimer's Disease and Parkinsons'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 & 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 tRNA Lys
MERRF
Encephalomyopathy
MTTK MERRF T8356C tRNA Lys
MERRF
Encephalomyopathy
MTTK MERRF G8361A tRNA Lys
MERRF
Encephalomyopathy MERRF/MICM+DEAF/Autis
MTTK G8363A tRNA Lys
MERRF m
Encephalomyopathy tRNA Leu
MTTL1 MERRF/KSS overlap G3255A
MERRF (UUR)
Encephalomyopathy
MTTF MERRF G611A tRNA Phe
MERRF
Encephalomyopathy
Myoclonus and tRNA
MTTD MEPR A7543G
Psychomotor Asp
Regression
Encephalomyopathy
Ataxia, Myoclonus MTTV AMDF G1606A tRNA Val
and Deafness
Encephalomyopathy MERRF-MELAS / Cerebral
MTTH G12147A tRNA His
MERRF edema
Encephalomyopathy tRNA Leu
MTTL1 MELAS A3243G
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS G3244A
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS A3252G
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS C3256T
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS/Myopathy T3258C
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS T3271C
MELAS (UUR)
Encephalomyopathy tRNA Leu
MTTL1 MELAS T3291C
MELAS (UUR)
Encephalomyopathy
MTTV MELAS G1642A tRNA Val
MELAS
Encephalomyopathy
MTTQ MELAS/Encephalopathy G4332A tRNA Gln
MELAS
Encephalomyopathy
MTTF MELAS G583A tRNA Phe
MELAS
Encephalomyopathy MTRNR 16S
MELAS C3093G
MELAS 2 rRNA
tRNA Leu
Encephalomyopathy MTTL1 PEM T3271delT
(UUR)
Encephalomyopathy MTTI Progressive Encephalopathy T4290C tRNA Ile
Mitochondrial
Encephalomyopathy MTTI C4320T tRNA Ile
Encephalocardiomyopathy
Encephalomyopathy MTTW Encephalomyopathy G5540A tRNA Trp
Encephalomyopathy MTTC Encephalopathy T5814C tRNA Cys
tRNA Ser
Encephalomyopathy MTTS1 PEM/AMDF C7472insC
(UCN)
tRNA Ser
Encephalomyopathy MTTS1 PEM/MERME T7512C
(UCN)
Encephalomyopathy MTTK Encephalopathy C8302T tRNA Lys
Encephalomyopathy MTTK Mitochondrial Encephalopathy G8328A tRNA Lys
Encephalomyopathy MTTG PEM T10010C tRNA Gly
Encephalomyopathy MTATT Encephalomyopathy G15915A tRNA Thr
Encephaolmyopathy MTRNR rRNA
Rett Syndrome C2835T
Rett Syndrome 2 16S
Multisystem Disease MTTI Varied familial presentation G4284A tRNA Ile
Encephalomyopathy
Gastrointestinal
Reflux and Sudden MTTG GER/SIDS A10044G tRNA Gly
Infant Death
Syndrome
Mitochondrial
MTTF MM T582C tRNA Phe
Myopathy
Mitochondrial
MTTF MM T618C tRNA Phe
Myopathy
Mitochondrial tRNA Leu
MTTL1 MM G3242A
Myopathy (UUR)
TRNA
Mitochondrial
MTTL1 MM/CPEO A3243G Leu(UUR
Myopathy
tRNA
Mitochondrial
MTTL1 MM A3243T Leu(UUR
Myopathy
Mitochondrial tRNA Leu
MTTL1 MM/CPEO T3250C
Myopathy (UUR)
Mitochondrial tRNA Leu
MTTL1 MM A3251G
Myopathy (UUR)
Mitochondrial tRNA Leu
MTTL1 MM C3254G
Myopathy (UUR)
Mitochondrial tRNA Leu
MTTL1 Myopathy A3280G
Myopathy (UUR)
TRNA
Mitochondrial
MTTL1 Myopathy A3288G Leu(UUR
Myopathy
Mitochondrial tRNA Leu
MTTL1 MM A3302G
Myopathy (UUR)
Mitochondrial
MTTI MM A4267G tRNA Ile
Myopathy
Mitochondrial
MTTQ Myopathy T4370AT tRNA Gln
Myopathy
Mitochondrial tRNA
MTTM MM T4409C
Myopathy Met
Mitochondrial tRNA
MTTM MM G4450A
Myopathy Met
Mitochondrial
MTTW MM G5521A tRNA Trp
Myopathy
Mitochondrial tRNA Ser
MTTS1 MM T7480G
Myopathy (UCN)
Mitochondrial tRNA Ser
MTTS1 MM G7497A
Myopathy (UCN)
Mitochondrial
MTTK Myopathy T8355C tRNA Lys
Myopathy
Mitochondrial
MTTK Myopathy T8362G tRNA Lys
Myopathy
Mitochondrial
MTTG Myopathy G10014A tRNA Gly
Myopathy
Mitochondrial tRNA Leu
MTTL2 MM A12320G
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
Myopathy, Exercise MTTY Exercise Intolerance T5874G tRNA Tyr
Intolerance
Mitochondrial tRNA Leu
MTTL1 CPEO C3254T
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
Mitochondrial tRNA
MTTN CPEO/MM T5692C
Myopathy, CPEO Asn
Mitochondrial tRNA
MTTN CPEO/MM G5698A
Myopathy, CPEO Asn
Mitochondrial tRNA
MTTN CPEO/MM G5703G
Myopathy, CPEO Asn
Mitochondrial
MTTK CPEO + Myoclonus G8342A tRNA Lys
Myopathy, CPEO
Mitochondrial tRNA Leu
MTTL2 CPEO G12294A
Myopathy, CPEO (CUN)
Mitochondrial tRNA Leu
MTTL2 CPEO/Stroke/CM A12308G
Myopathy, CPEO (CUN)
Mitochondrial tRNA Leu
MTTL2 CPEO T12311C
Myopathy, CPEO (CUN)
Mitochondrial tRNA Leu
MTTL2 CPEO G12315A
Myopathy, CPEO (CUN)
Mitochondrial
tRNA Leu
Myopathy, Ocular MTTL1 Ocular myopathy T3273C
(UUR)
Myopathy
Mitochondrial tRNA Leu
MTTL1 KSS G3249A
Myopathy, KSS (UUR)
Mitochondrial
Mitochondrial Cytopathy/
Myopathy MTTY A5843G tRNA Tyr
FSGS
Cytopathy
Mitochondrial
Myopathy MTTK Mitochondrial cytopathy A8326G tRNA Lys
Cytopathy
Mitochondrial
Myopathy MTTP Mitochondrial cytopathy G15995A tRNA Pro
Cytopathy
Mitochondrial
TRNA
Myopathy with MTTF Myoglobinuria A606G
Myoglobinuria
Mitochondrial
Myopathy,
MTTW Gastrointestinal Syndrome G5532A tRNA Trp
Gastrointestinal
Syndrome
Mitochondrial
Myopathy,
Mitochondrial
MTTK MNGIE G8313A tRNA Lys
Neurogastrointestina
Encephalomyopathy
Mitochondrial
Myopathy with
MTTG CIPO A10006G tRNA Gly
Chronic Intestinal
Pseudoobstruction
Mitochondrial
Myopathy with tRNA Ser
MTTS1 CIPO C12246G
Chronic Intestinal (AGY)
Pseudoobstruction
Mitochondrial
Myopathy with MTTF Tubulointerstitial nephritis A608G tRNA Phe
Renal Dysfunction
Mitochondrial
Myopathy Lethal
MTTT LIMM A15923G tRNA Thr
Infantile
Mitochondrial
Myopathy
Mitochondrial
Myopathy Lethal
Infantile MTTT LIMM A15924G tRNA Thr
Mitochondrial
Myopathy
Mitochondrial
tRNA Leu
Myopathy and MTTL1 MMC A3260G
(UUR)
cardiomyopathy
Mitochondrial
tRNA Leu
Myopathy and MTTL1 MMC C3303T
(UUR)
cardiomyopathy
Maternaly Inherited
Hypertrophic MTTI MHCM A4295G tRNA Ile
Cardiomyopathy
Maternally Inherited
MTTI MICM A4300G tRNA Ile
Cardiomyopathy
Cardiomyopathy MTTK Cardiomyopathy A8348G tRNA Lys
Maternally Inherited
Hypertrophic MTTG MHCM T9997C tRNA Gly
Cardiomyopathy
Maternally Inherited
MTTH MICM G12192A tRNA His
Cardiomyopathy
tRNA Leu
Cardiomyopathy MTTL2 Dilated Cardiomyopathy T12297C
(CUN)
Fatal Infantile
Cardiomyopathy MTTI FICP A4269G tRNA Ile
Plus (MELAS)
Fatal Infantile
Cardiomyopathy MTTI FICP A4317G tRNA Ile
Plus (MELAS)
MTRNR 12S
Deafness DEAF A827G
1 rRNA
MTRNR 12S
Deafness DEAF T961C
1 rRNA
MTRNR T961delT+C(n)in 12S
Deafness DEAF
1 s rRNA
MTRNR 12S
Deafness DEAF T961insC
1 rRNA
MTRNR 12S
Deafness DEAF T1005C
1 rRNA
Deafness
MTRNR 12S
Sensory Neural SNHL T1095C
1 rRNA
Hearing Loss
MTRNR 12S
Deafness DEAF A1116G
1 rRNA
MTRNR 12S
Deafness DEAF C1494T
1 rRNA
MTRNR 12S
Deafness DEAF A1555G
1 rRNA
Deafness
tRNA Ser
Sensory Neural MTTS1 SNHL T7510C
(UCN)
Hearing Loss
Deafness
tRNA
Sensory Neural MTTS1 SNHL T7511C
Ser(UCN)
Hearing Loss
Deafness
Deafness and Cerebellar tRNA
cerebellar MTTS1 7472insC
Dysfunction Ser(UCN)
dysfunction
Deafness MTTH DEAF + RP G12183A tRNA His
Deafness Ataxia and Deafness, Mental Retaration,
MTTE 14709G tRNA Glu
MR Cerebellar Dysfunction
MTRNR
Diabetes Mellitus DM C1310T 12S
MTRNR
Diabetes Mellitus DM A1438G 12S
Diabetes Mellitus & tRNA Leu
MTTL1 DM / DMDF A3243G
Deafness (UUR)
tRNALeu
Diabetes Mellitus MTTL1 DM T3264C
(UUR)
tRNA Leu
Diabetes Mellitus MTTL1 DM T3271C
(UUR)
Diabetes Mellitus
Metabolic Syndrome &
MTTI T4291C tRNA Ile
Hypomagnesemia
Metabolic
Syndrome
Diabetes Mellitus &
Deafness & MTTK DMDF/MERRF/HCM A8296G tRNA Lys
Cardiomyopathy
Diabetes Mellitus &
tRNA Ser
Deafness and MTTS2 DMDF C12258A
(AGY)
Retinitis Pigmentosa
Movement Disorder MTTV Movement Disorder T1659C tRNA Val
Alzheimer & MTRNR rRNA
ADPD G3196A
Parkinson Disease 2 16S
Alzheimer &
Parkinson Disease ADPD/Hearing loss and
MTTQ T4336C tRNA Gln
Deafness & migraine
Migraine
Dementia and
MTTW DEMCHO G5549A tRNA Trp
Chorea
Abbreviations
Plasmy: Ho, homoplasmy; He, heteroplasmy
* Disease: AD, Alzheimer's Disease; ADPD, Alzheimer's Disease and Parkinsons'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 & 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.
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 Sudden Infant Brain
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
WHAT I/WE
Claims (27)
1. An isolated codon optimised nucleic acid sequence encoding the yeast NDI1 protein of SEQ ID NO: 542 or a sequence having at least 95% sequence identity with SEQ ID NO: 542, that, compared with the sequence of the wild-type yeast NDI1 gene of SEQ ID NO: 1, comprises at least 50 codons that are codon optimised for expression in mammalian cells.
2. The nucleic acid sequence of Claim 1, comprising at least 100, 200, 300 or 329 codons which are codon optimised compared with the sequence of wild-type yeast NDI1 gene of SEQ ID NO: 1.
3. The nucleic acid sequence of either of claims 1 and 2, wherein said NDI1 protein is immune optimised.
4. The nucleic acid sequence of claim 3, wherein said isolated nucleic acid sequence encodes an immune optimised functional variant of the yeast NDI1 protein of SEQ ID NO: 542 comprising at least one conservative amino acid change to a residue selected from the group consisting of: L19, L150, L151, L195, and L259.
5. A nucleic acid construct comprising the nucleic acid sequence of any one of the preceding claims and a nucleic acid sequence encoding a mitochondrial localisation sequence.
6. A vector suitable for use in gene therapy and comprising a nucleic acid sequence of any one of Claims 1 to 4.
7. The vector of Claim 6, in which the vector is an adeno-associated virus (AAV).
8. The vector of Claim 6 or 7, comprising a nucleic acid sequence encoding a mitochondrial localisation sequence.
9. The vector as claimed in any one of Claims 6 to 8, for intraocular delivery.
10. The vector as claimed in any one of Claim 6 to 9, and additionally comprising the GDNF gene.
11. The vector as claimed in any one of Claims 6 to 9, and additionally comprising a gene that enhances cell survival and or cell function.
12. The vector of Claim 11, wherein the gene that enhances cell survival or cell function is selected from a gene encoding a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, or a hormone.
13. The vector as claimed in any one of Claims 6 to 12, comprising a promotor, wherein the at least one nucleic acid is expressed from the promotor.
14. The vector as claimed in Claim 13 in which the promotor is one that is preferentially or specifically expressed in retinal ganglion cells (RGC’s) wherein expression of the nucleic acid is under the control of the promotor.
15. A vector suitable for use in transfecting a mammalian retinal ganglion cell and comprising a nucleic acid according to any one of Claims 1 to 4 and a nucleic acid encoding GDNF, in which the vector is an AAV virus.
16. A pharmaceutical formulation suitable for intraocular delivery and comprising the vector of any one of Claims 6 to 15.
17. A system comprising a vector of any one of Claims 6 to 15 in combination with a second vector comprising the GDNF gene.
18. A system comprising a vector of any one of Claims 6 to 15 in combination with a second vector comprising a gene that enhances cell survival and or cell function.
19. The system of Claim 18 in which the second vector comprises a gene encoding a neurotrophic factor, a growth factor, an anti-apoptotic agent, an antioxidant, a cytokine, or a hormone.
20. An isolated cell transformed with a nucleic acid of any one of Claims 1 to 4 or the vector of any one of Claims 6 to 15 or the system of any one of Claims 17 to 19.
21. The cell as claimed in Claim 20 selected from a stem cell, progenitor cell, RGC, or RGC precursor cell.
22. A nucleic acid of any one of Claims 1 to 4, a construct of Claim 5, a vector of any one of Claims 6 to 15, the formulation of Claim 16, the system of any one of Claims 17 to 19, or the cell of Claims 20 or 21, for use as a medicament.
23. A nucleic acid of any one of Claims 1 to 4, a construct of Claim 5, a vector of any one of Claims 6 to 15, the formulation of Claim 16, the system of any one of Claims 17 to 19, or the cell of Claims 20 or 21, for use in the treatment of a disease or condition associated with mitochondrial dysfunction.
24. The nucleic acid, construct, vector, formulation, system, or cell for use according to Claim 23, wherein the disease or condition associated with mitochondrial dysfunction is selected from the group consisting of a neurodegenerative disease, a muscular disease and Leber Hereditory Optic Neuropathy (LHON).
25. Use of a nucleic acid of any one of Claims 1 to 4, a construct of Claim 5, a vector of any one of Claims 6 to 15, the formulation of Claim 16, the system of any one of Claims 17 to 19, or the cell of Claims 20 or 21, in the manufacture of a medicament.
26. Use of a nucleic acid of any one of Claims 1 to 4, a construct of Claim 5, a vector of any one of Claims 6 to 15, the formulation of Claim 16, the system of any one of Claims 17 to 19, or the cell of Claims 20 or 21, in the manufacture of a medicament for treating a disease or condition associated with mitochondrial dysfunction.
27. Use of claim 26, wherein the disease or condition associated with mitochondrial dysfunction is selected from the group consisting of a neurodegenerative disease, a muscular disease and Leber Hereditory Optic Neuropathy (LHON).
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11194796.6A EP2607375A1 (en) | 2011-12-21 | 2011-12-21 | Variants of yeast NDI1 gene, and uses thereof in the treatment of disease associated with mitochondrial dysfunction. |
EP11194796.6 | 2011-12-21 | ||
NZ627287A NZ627287B2 (en) | 2011-12-21 | 2012-12-21 | Variants of yeast ndi1 gene, and uses thereof in the treatment of disease associated with mitochondrial dysfunction |
Publications (2)
Publication Number | Publication Date |
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NZ724376A NZ724376A (en) | 2019-09-27 |
NZ724376B2 true NZ724376B2 (en) | 2020-01-07 |
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