NZ736083B2 - Oligonucleotides matching col7a1 exon 73 for epidermolysis bullosa therapy - Google Patents
Oligonucleotides matching col7a1 exon 73 for epidermolysis bullosa therapy Download PDFInfo
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- NZ736083B2 NZ736083B2 NZ736083A NZ73608316A NZ736083B2 NZ 736083 B2 NZ736083 B2 NZ 736083B2 NZ 736083 A NZ736083 A NZ 736083A NZ 73608316 A NZ73608316 A NZ 73608316A NZ 736083 B2 NZ736083 B2 NZ 736083B2
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- New Zealand
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- exon
- oligoribonucleotide
- antisense
- mrna
- oligonucleotide
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Classifications
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- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- 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
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- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C—CHEMISTRY; METALLURGY
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- C12N2320/33—Alteration of splicing
Abstract
Antisense oligonucleotides capable of preventing or reducing exon 73 inclusion into the human COL7A mRNA are characterized in various ways: (a) the oligonucleotide's sequence includes at most two CpG sequences; (b) the oligonucleotide has a length of no more than 24 nucleotides; (c) the oligonucleotide is capable of annealing to the (SRp40/SC35 binding / ESE) element in exon73. These oligonucleotides can usefully be oligoribonucleotides with modified internucleosidic linkages e.g. phosphorothioate linkages. ide is capable of annealing to the (SRp40/SC35 binding / ESE) element in exon73. These oligonucleotides can usefully be oligoribonucleotides with modified internucleosidic linkages e.g. phosphorothioate linkages.
Description
(12) Granted patent specificaon (19) NZ (11) 736083 (13) B2
(47) Publicaon date: 2021.12.24
(54) OLIGONUCLEOTIDES MATCHING COL7A1 EXON 73 FOR EPIDERMOLYSIS BULLOSA THERAPY
(51) Internaonal Patent Classificaon(s):
C12N 15/113 A61K 31/712 A61K 31/7125
(22) Filing date: (73) Owner(s):
2016.03.11 Wings Therapeutics, Inc.
(23) Complete specificaon filing date: (74) Contact:
2016.03.11 Jones Tulloch
(30) Internaonal Priority Data: (72) Inventor(s):
GB 1504124.7 2015.03.11 HAISMA, Elisabeth, Marlene
POTMAN, Marko
(86) Internaonal Applicaon No.: BEUMER, Wouter
BRINKS, Vera
(87) Internaonal Publicaon number:
WO/2016/142538
(57) Abstract:
Ansense oligonucleodes capable of prevenng or reducing exon 73 inclusion into the human
COL7A mRNA are characterized in various ways: (a) the oligonucleode's sequence includes at
most two CpG sequences; (b) the oligonucleode has a length of no more than 24 nucleodes; (c)
the oligonucleode is capable of annealing to the (SRp40/SC35 binding / ESE) element in exon73.
These oligonucleodes can usefully be oligoribonucleodes with modified internucleosidic linkages
e.g. phosphorothioate linkages.
NZ 736083 B2
OLIGONUCLEOTIDES MATCHING COL7A1 EXON 73 FOR EPIDERMOLYSIS BULLOSA THERAPY
This application claims the benefit of United Kingdom patent application 1504124.7, filed 11th March
2015, the complete contents of which are hereby incorporated herein by reference for all purposes.
FIELD OF THE INVENTION
The present invention is concerned with oligonucleotides suitable for use in treating human disease.
More in particular the present invention is concerned with antisense oligonucleotides suitable for the
treatment of dystrophic epidermolysis bullosa.
DISEASE BACKGROUND
Epidermolysis Bullosa (EB) is a group of heritable skin diseases, which are characterized by chronic
fragility and blistering of the skin and mucous membranes. Depending on the subtype, the spectrum of
symptoms of the EB is very broad, ranging from minimal skin fragility to very severe symptoms with
general complications. Worldwide about 350,000 patients are affected. In some forms of EB, also nails,
hair and teeth may be involved. The main types of EB include EB Simplex (EBS), Junctional EB (JEB),
Dystrophic EB (DEB) and Kindler syndrome (KS).
DEB affects about 25% of EB patients, can be either dominantly or recessively inherited, and involves
defects in Type VII collagen (COL7A1, omim 120120). COL7A1 encoding the alpha-1 chain of collagen VII.
Collagen VII functions as an anchoring fibril of the upper part of the dermis to the lamina densa (part of
the basement membrane). Following post-translational modification three identical alpha-1 chains fold
together with their collagenous triple helix domain. Subsequently, antiparallel dimers are formed that
align to form the anchoring fibrils. Collagen VII is synthesized in the skin by keratinocytes and dermal
fibroblasts. DEB disease severity roughly correlates with the amount of type VII collagen expression at
the basement membrane zone.
Characteristics of Dominant Dystrophic EB (DDEB) include blistering that may be localized to the hands,
feet, elbows and knees or generalized. Common findings include scarring, milia, mucous membrane
involvement, and abnormal or absent nails. Recessive Dystrophic EB (RDEB) is typically more generalized
and severe than DDEB. In addition to the findings of DDEB, other common manifestations of RDEB
include malnutrition, anemia, osteoporosis, esophageal strictures, growth retardation, webbing, or
fusion of the fingers and toes causing mitten deformity (pseudosyndactyly), development of muscle
contractures, malformation of teeth, microstomia and scarring of the eye. The risk of squamous cell
carcinoma is greatly increased in this group as well as death from metastatic squamous cell carcinoma.
Within the gene COL7A1 more than 400 different mutations are known. One of the most prevalent
affected exons (18% of patients) is exon 73 with about 40 known mutations, most often missense
mutations or mutations leading to premature termination codons (PTCs) and glycine substitutions.
Currently there is no treatment for DEB, only palliative care is performed. Severe forms of RDEB impose
a high cost on society’s healthcare budget: the average costs of dressings and medication is about €
200,000 per patient per year.
WO2013/053819 of Institut National de la Santé et de la Recherche Médicale (INSERM) discloses two
antisense oligonucleotides targeting exon 73, causing the entire exon to be skipped from the mRNA. The
exondeficient mRNA is translated into a functional polypeptide that, although being shorter than the
40 wt protein, behaves very similar to wild-type collagen VIIa. One oligonucleotide disclosed is 25
nucleotides in length, displaying a skipping efficiency of 69%, while the other is 30 nucleotides in length,
displaying 93% skipping efficiency.
SUMMARY OF THE INVENTION
Although the longer exon-skipping AON in WO2013/053819 appears to display satisfactory exon
skipping efficiency, its length and some other characteristics make it less preferred from the perspective
of developing such a molecule for human therapeutic use. Besides, it appears that this oligonucleotide
produces intermediate bands that are neither representative of wild-type, nor of exon 73-free mRNAs.
Although it is not known whether these bands have clinical relevance, producing by-products is less
preferred from a regulatory and safety standpoint. Hence, there remains a need for further and
improved therapies to treat DEB.
Thus the invention provides an antisense oligonucleotide capable of preventing or reducing exon 73
inclusion into the human COL7A1 mRNA, when said mRNA is produced by splicing from a pre-mRNA in a
mammalian cell; characterized in that (a) the oligonucleotide’s sequence includes at most two CpG
sequences and/or (b) the oligonucleotide has a length of no more than 24 nucleotides. Advantageously,
the oligonucleotide has both properties (a) and (b).
The invention also provides an antisense oligonucleotide capable of preventing or reducing exon 73
inclusion into the human COL7A1 mRNA, when said mRNA is produced by splicing from a pre-mRNA in a
mammalian cell, characterized in that the oligonucleotide is capable of annealing to the (SRp40/SC35
binding / ESE) element in exon 73 characterized by the sequence 5'-UUUCCUGG-3' (SEQ ID NO: 4). This
oligonucleotide can have properties (a) and/or (b) as discussed above.
In a preferred embodiment, the present invention provides an antisense oligoribonucleotide capable of
preventing or reducing exon 73 inclusion into the human COL7A1 mRNA, when said mRNA is produced
by splicing from a pre-mRNA in a mammalian cell, characterized in that the oligoribonucleotide is
capable of annealing to the (SRp40/SC35 binding / ESE) element in exon 73 characterized by the
sequence 5’-UUUCCUGG-3’ (SEQ ID NO: 4), wherein the oligoribonucleotide has a length of between 16
and 24 nucleotides.
In another preferred embodiment, the present invention provides an antisense oligoribonucleotide
capable of preventing or reducing exon 73 inclusion into the human COL7A1 mRNA, when said mRNA is
produced by splicing from a pre-mRNA in a mammalian cell, characterized in that the
oligoribonucleotide is selected from the group consisting of the antisense oligoribonucleotides (AONs)
of SEQ ID NO: 5, 6, 7, 8, 24, 25, 26, 27, 28, 39, 40, 41, 42, 43, 29 and 35.
Oligonucleotides of the invention can usefully be oligoribonucleotides with modified internucleosidic
linkages e.g. phosphorothioate linkages. They can also have modified sugars e.g. with 2'-O-methyl
substituted sugar moieties. These and other details of the oligonucleotides are discussed below.
DESCRIPTION OF THE FIGURES
Figure 1 shows the human Col7A1 exon 73 (SEQ ID NO: 1; upper case) with its 5’ and 3’ flanking intron
boundaries (SEQ ID NOs: 2 & 3; lower case).
Figure 2 shows the location of SR protein binding sites in exon 73 and the location of AONs.
Figure 3 shows lab-on-a-chip results for exon skipping on primary human fibroblasts (HPF) cells. The full-
length mRNA gives a band at ~350 bp, whereas mRNA with excluded exon 73 is ~150 bp.
Figure 4 shows the histological results of delivery of mh-AON1 formulated in PBS using an ex vivo
40 porcine skin model. 4A-4B show the results of having 25 µg of mh-AON on intact skin for 24 hours, 4C-4F
show the results of having 25 µg of mh-AON1 on blister-like skin, with the complete epidermis removed.
C-D: incubation for 24 hours. E-F: incubation for 48 hours. mh-AON1 is stained (red). Scale bar is 100
Figure 5 shows the histological results of delivery of mh-AON1 formulated into three different hydrogels
using the same ex vivo porcine skin model as for Figure 4. 5A-5B shows the results of porcine skin
treated with saline controls (A) with intact epidermis and (B) with removed epidermis. 5C-5D shows the
porcine skin treated with 50 µg mh-AON1-cy5 mixed in Flaminal™, (C) with intact epidermis (D) with
epidermis removed. 5E-5F shows the results with porcine skin treated with 50 µg mh-AON1-cy5 mixed in
carbomer hydrogel (E) with intact epidermis and (F) with epidermis removed. 5G-5H shows the results
with porcine skin treated with 50 µg mh-AON1-cy5 mixed in hypromellose hydrogel (G) intact skin and
(H) with removed epidermis. Scale bar indicates 100 µm. mh-AON1 is stained (red).
Figure 6 shows lab-on-a-chip results of splicing products of COL7A1 mRNA after treatment with mh-
AON1 or the scrambled variant (SCRM) as a control oligo. Two different cell types we tested (HeLa and
HPF), both with 100nM oligonucleotide for either 24 h (left four lanes) or 40 h (right 4 lanes). Different
COL7A1 mRNA products are formed after treatment with mh-AON1 or control oligo (including and
excluding exon 73). The different mRNA products were analysed for length; 350 fragment represents the
wild type, full length, mRNA and the 150 nucleotide fragment the modulated mRNA product.
Figure 7 shows primer design for the ddPCR assay. Two different primer combinations were designed to
PCR either only the wild type product (top) or the Δexon 73 product (bottom). Upper row: primer pair
for the wild type; Lower row: primer pair for the skipped exon 73.
Figure 8 shows the absolute quantification (% of total copies; y-axis) of COL7A1 mRNA transcripts
including exon 73 and excluding exon 73, in HPF cells that carry an unaltered COL7A1 sequence. A dose-
response was done with mh-AON1, with 50, 100 and 200 nM (x-axis). Results are shown 24 hours (left)
or 40 hours (right) after transfection with the oligonucleotide. Black bars represent the full length
product while the grey bars represent the transcript Δ73.
Figure 9 shows the results of the immunogenicity and immunotoxicity assessment of mh-AON1 in
human PBMC. (a) Heat map depicting the significance levels of cytokine concentrations in culture
supernatant after 24 h stimulation of human PBMC with mh-AON1 (10nM, 100nM or 1µM) or the
positive controls Poly(I:C) (1µg/ml), CpG (10µg/ml), LPS (100ng/ml) and R848 (1µM) compared to saline-
treated human PBMC. Every square shows the reached significance level per treatment condition
(geometric mean of the five human donor with triplicate measurements each) for each measured
cytokine. (b) Fold change of IFN-α2 concentration in culture supernatant after 24hrs stimulation of
PBMC with mh-AON1 or the positive controls compared to saline-treated PBMC. Bars depict the mean
with SEM of triplicate measurements per human donor (in different grey tones). The dotted line at 1
depicts the relative cytokine concentration of the saline treated PMBCs. P-values in (a) and (b) were
determined using the Friedman test with Dunn’s post-hoc test (c) Relative number of viable PBMC
expressed as fold change of Resorufin fluorescence compared to saline treated PBMC after 24 h
exposure to mh-AON1 or the positive controls. Viable cell assessment was performed using the
CellTiter-Blue kit. For all individual biological replicates, fold changes were calculated by normalizing
40 measured RFU against geometric mean of corresponding triplicate saline control. Results are shown per
individual donor as the mean+SEM of the triplicate fold change, normalized against the mean of its
corresponding saline control (dotted line). Repeated measures One-way ANOVA with Dunnett test for
multiple corrections (compared to saline) was performed. (*P<0.05, **P ≤ 0.01, ****P < 0.001).
Figure 10 shows the results of the immunogenicity and immunotoxicity assessment of mh-AON1 and
AON73.24.5 in human Ramos-Blue cells. (a) NF-kB/AP-1 activation in Ramos-Blue cells after 24 h
incubation with mh-AON1 or AON73.24.5 (at several concentrations) and the TLR agonists Poly(I:C)
(1µg/ml), CpG (10µg/ml), LPS (100ng/ml) and R848 (1µM). (b) Relative number of viable Ramos-Blue
cells expressed as fold change of resorufin fluorescence compared to saline treated Ramos-Blue cells
after 24hrs exposure to mh-AON1, AON73.45.5 or the positive controls. Viable cell assessment was
performed using the CellTiter-Blue kit. For all individual biological replicates, fold changes were
calculated by normalizing measured O.D (in a) or RFU (in b) against geometric mean of corresponding
triplicate saline control. Results are shown per as the mean+SEM of the triplicate fold change,
normalized against the mean of its corresponding saline control (dotted line). Repeated measures One-
way ANOVA with Dunnett test for multiple corrections (compared to saline) was performed on the fold
change values. (****P < .0001).
DETAILED DESCRIPTION OF THE INVENTION
Surprisingly, it has now been found by the inventors that antisense oligonucleotides can be designed
that fulfill the requirements for an AON to develop them into therapeutics to treat human disease, in
particular dystrophic epidermolysis bullosa (DEB).
Although AON 73.3 disclosed in WO2013/053819 appears to be satisfactory in terms of reducing exon
73 inclusion in the COL7A1 mRNA, this oligonucleotide is unnecessarily long with its length of 30nt,
which is less preferred from a manufacturability, CMC and cost of goods point of view. Moreover, the
INSERM oligonucleotides contain multiple CpG repeats, which is less preferred from an immunogenicity
standpoint. It is known that CpGs, especially repeats thereof, interact with the TLR9 receptor, thereby
causing an immune response in the treated individual which may harm performance and/or cause harm
to the tissues treated with the oligonucleotide.
Preferred AONs of the invention are less than 25, preferably less than 24, nucleotides in length, capable
of preventing, or at least reducing, inclusion of exon 73 into the COL7A1 mRNA with high efficiency and,
compared to the prior art, have fewer (and preferably no) structures or sequences that might hamper
functionality.
The shortened mRNA, lacking the entire exon 73 as a result of treatment using AONs of the invention,
will be translated into a shorter but functional COL VII protein.
The AONs of the invention preferably contain no more than two (preferably only one, or even none)
CpG sequence(s) and/or range in length between 16 and 24 nucleotides, while achieving exon skipping
efficiencies of more than 60% (e.g. more than 70%, ideally more than 75% or 80%, preferably more than
85%, and still more preferably more than 90%) as measured in HeLa cells.
In a different aspect of the invention, AONs have been designed capable of annealing to an 8-mer motif
that was, until now, not recognized as being important in selection of the 5’ splice acceptor site flanking
exon 73. It is postulated that this 8-mer motif is a previously overlooked exonic splicing enhancer (ESE)
that may be targeted to prevent, or at least reduce, exon 73 inclusion into the COL7A1 mRNA. The
inventors used a microwalk technique to determine the location of this newly recognized putative ESE,
using AONs capable of annealing to the entire motif or part of the motif, designing different AONs that
40 are progressively truncated to shorten the overlap with this motif until exon skipping is lost entirely. By
so doing, the inventors identified a 5'-UUUCCUGG-3' motif (SEQ ID NO: 4) in the 5'-region of exon 73
(see Figure 1) that forms an excellent new target for AONs to bring about prevention, or at least
reduction, of exon 73 inclusion into the COL7A1 mRNA.
In a further embodiment of the invention, an AON is disclosed which is capable of efficiently preventing,
or at least reducing, exon 73 inclusion in the COL7A1 mRNA in both mice and humans. This AON (m-h
AON1) is fully complementary to the pre-mRNA target in both mice and humans. This AON has as
advantage that it can be used to perform proof of concept studies and toxicology studies in mice using
the exact same molecule as the one that will eventually be developed for therapeutic use in humans.
None of the AONs according to the invention appear to produce intermediate bands; only bands
corresponding to the wt mRNA or bands corresponding to a full exon 73 less mRNA appear to be
generated in cells treated with AONs according to the invention.
A further preferred property of AONs according to the invention is that they do not contain G-tetrads or
multiple G’s (3 or more consecutive guanosines), thereby avoiding problems associated with multiplex
formation and/or solubility.
Table 1 shows for each AON the skipping efficiency of exon 73 in HeLa cells, the nucleotide sequence
and SEQ ID NO of preferred AONs according to the invention (AON1 – AON25 and m-hAON1), of the
AONs used in the micro-walk to identify the new ESE-motif (AON26-30), and of truncated versions of the
AONs found to bind this ESE-motif which lack undesirable structures such as G-tetrads (AON24.1 to
24.5) while still displaying satisfactory exon skipping efficiencies. Further details about the AONs, their
efficacy in other cells, and comparison to prior art AONs, are given in Example 1.
Table 1: Efficiency of exon 73 exclusion from mRNA. HPF and HeLa cells were treated for 24
hours with 100nm AON.
AON sequence 5’ – 3’ SEQ ID NO
HeLa
86% 5
UCUCCAGGAAAGCCGAUGGGGCCC
AON1
85% AGCCCGCGUUCUCCAGGAAAGCCGA 6
AON2
92% 7
GUCGCCCUUCAGCCCGCGUUCUCCA
AON3
83% ACGGUCGCCCUUCAGCCCGCGUU 8
AON4
3% 9
CCCCUGAGGGCCAGGGUCUCCACGG
AON5
0% CAGACCAGGUGGCCCCUGAGGGCCA 10
AON6
CCAAGGGCCAGACCAGGUGGCCCC 11
AON7 0%
CCAGACCAGGUGGCCCCUGAGGGCC 12
AON8 0%
UCUCCCCAAGGGCCAGACCAGG 13
AON9 0%
GGAAGGCCCGGGGGGGCCCCUCUC 14
AON10 0%
CCGGCAAGGCCGGAAGGCCCGGGG 15
AON11 6%
AGGCUUUCCAGGCUCCCCGGCAAG 16
AON12 0%
CGGGAAUACCAGGCUUUCCAGGCU 17
AON13 2%
UGCCUGGGAGCCCGGGAAUACCA 18
AON14 25%
CCCACACCCCCAGCCCUGCCUGGG 19
AON15 8%
CCUCUCCCACACCCCCAGCCCU 20
AON16 0%
UCUCUCCUGGCCUUCCUGCCUCU 21
AON17 9%
CACCCUCUCUCCUGGCCUUCCU 22
AON18 13%
CCAGCCUCACCCUCUCUCCUGG 23
AON19 7%
CUCCAGGAAAGCCGAUGGGGCCC 24
AON20 100%
UCCAGGAAAGCCGAUGGGGCCC 25
AON21 89%
CCAGGAAAGCCGAUGGGGCCC 26
AON22 85%
CUCCAGGAAAUCCGAUGGGGCCcu 27
AON23 83%
UCCAGGAAAGCCGAUGGGGCCcug 28
AON24 93%
AON24.1 73% UCCAGGAAAGCCGAUGGG
UCCAGGAAAGCCGAUGG 40
AON24.2 88%
UCCAGGAAAGCCGAUG 41
AON24.3 79%
CUCCAGGAAAGCCGAUGG 42
AON24.4 86%
UCUCCAGGAAAGCCGAUG 43
AON24.5 89%
CCAGGAAAGCCGAUGGGGCCcugc 29
AON25 92%
AGGAAAGCCGAUGGGGCCcugcag 30
AON26 49%
GAAAGCCGAUGGGGCCcugcagga 31
AON27 37%
AAGCCGAUGGGGCCcugcaggagu 32
AON28 47%
GCCGAUGGGGCCcugcaggagugg 33
AON29 0%
GAUGGGGCCcugcaggaguggaa 34
AON30 7%
CGUUCUCCAGGAAAGCCGAUG 35
mh-AON 1 91%
According to one embodiment, an antisense oligonucleotide is provided that is capable of preventing or
reducing exon 73 inclusion into the mammalian (preferably human) COL7A1 mRNA, when said mRNA is
produced by splicing from a pre-mRNA in a mammalian cell characterized in that the oligonucleotide’s
sequence has at least one of properties (a) and/or (b): (a) it includes at most two CpG sequences; and/or
(b) it has a length of no more than 24 nucleotides. For property (a), the oligonucleotide preferably
includes no more than one CpG sequence, and may include only one.
According to another embodiment, an antisense oligonucleotide is provided that is capable of
preventing or reducing exon 73 inclusion into the mammalian (preferably human) COL7A1 mRNA, when
said mRNA is produced by splicing from a pre-mRNA in a mammalian cell, characterized in that the
oligonucleotide is capable of annealing to the sequence motif 5'-UUUCCUGG-3' (SEQ ID NO: 4) in the
' upstream part of exon 73 (fig. 1). Without wishing to be bound by theory, this motif is postulated to
represent a SRp40/SC35 binding exonic splicing enhancer (ESE) element. The AONs according to this
embodiment are preferably characterized in that the oligonucleotide’s sequence has one or both of
properties (a) and/or (b) as discussed above. In order to have optimal effect the oligonucleotide should
anneal to the entire 8-mer motif; if exon skipping efficiencies below 60% would be acceptable for any
particular scenario then annealing to the 6 or 7 most 5' nucleotides of the 8-mer motif can be
acceptable.
Further preferred AONs according to the invention are those wherein feature (a) is characterized by that
the oligonucleotide includes no more than one CpG, and/or feature (b) is characterized in that the
oligonucleotide has a length of no more than 24 nucleotides, preferably between 12 and 24 nucleotides,
more preferably between 16 and 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides
still more preferably less than 23 nucleotides, still more preferably between 16 and 23 nucleotides, such
as 16, 17, 18, 19, 20, 21, 22, 23 nucleotides. According to most preferred embodiments of the invention,
the oligonucleotides are characterized in that they have both properties (a) at most two CpG sequences,
preferably no more than one, such as one CpG and (b) a length of no more than 24 nucleotides,
preferably between 12 and 24 nucleotides, more preferably between 16 and 24 nucleotides, such as 16,
17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, still more preferably less than 23 nucleotides, still more
preferably between 16 and 23 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 nucleotides.
An optional further feature of AONs according to the invention is that their sequence lacks a stretch of 3
or more consecutive guanosines.
Specific preferred AONs of the invention have the nucleotide sequences AON1, AON2, AON3, AON4,
AON20, AON21, AON22, AON23, AON24, AON24.1, AON24.2, AON24.3, AON24.3, AON.24.4, AON.24.5,
AON25 and mh-AON1 as disclosed in Table 1 above. More preferably for these oligos, all ribose moieties
are 2'-O-methylated and substantially all internucleosidic linkages are phosphorothioates.
In all embodiments of the present invention, the terms “preventing, or at least reducing, exon inclusion”
and “exon skipping” are synonymous. In respect of COL7A1, “preventing, or at least reducing, exon
inclusion” or “exon skipping” are to be construed as the exclusion of exon 73 (SEQ ID NO: 1, or allelic
forms thereof) from the human COL7A1 mRNA (see figure 1). The term exon skipping is herein defined
as the induction within a cell of a mature mRNA that does not contain a particular exon that would be
present in the mature mRNA without exon skipping. Exon skipping is achieved by providing a cell
expressing the pre-mRNA of said mature mRNA with a molecule capable of interfering with sequences
such as, for example, the splice donor or splice acceptor sequence required for allowing the biochemical
process of splicing, or with a molecule that is capable of interfering with an exon inclusion signal
required for recognition of a stretch of nucleotides as an exon to be included in the mature mRNA; such
molecules are herein referred to as exon skipping molecules.
The term pre-mRNA refers to a non-processed or partly-processed precursor mRNA that is synthesized
from a DNA template in a cell by transcription.
The term “antisense oligonucleotide” is understood to refer to a nucleotide sequence which is
complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogeneous
nuclear RNA) or mRNA molecule, so that it is capable of annealing with its corresponding target
sequence.
The term “complementary” as used herein includes “fully complementary” and “substantially
complementary”, meaning there will usually be a degree of complementarity between the
oligonucleotide and its corresponding target sequence of more than 80%, preferably more than 85%,
still more preferably more than 90%, most preferably more than 95%. For example, for an
oligonucleotide of 20 nucleotides in length with one mismatch between its sequence and its target
sequence, the degree of complementarity is 95%.
The degree of complementarity of the antisense sequence is preferably such that a molecule comprising
the antisense sequence can anneal to the target nucleotide sequence in the RNA molecule under
physiological conditions, thereby facilitating exon skipping. It is well known to a person having ordinary
skill in the art, that certain mismatches are more permissible than others, because certain mismatches
have less effect on the strength of binding, as expressed in terms of melting temperature or Tm,
between AON and target sequence, than others. Certain non-complementary base pairs may form so-
called “wobbles” that disrupt the overall binding to a lesser extent than true mismatches. The length of
the AON also plays a role in the strength of binding, longer AONs having higher melting temperatures as
a rule than shorter AONs, and the G/C content of an oligonucleotide is also a factor that determines the
strength of binding, the higher the G/C content the higher the melting temperature for any given length.
Certain chemical modifications of the nucleobases or the sugar-phosphate backbone, as contemplated
by the present invention, may also influence the strength of binding, such that the degree of
complementarity is only one factor to be taken into account when designing an oligonucleotide
according to the invention.
40 The presence of a CpG or multitude (two or more) of CpGs in an oligonucleotide is usually associated
with an increased immunogenicity of said oligonucleotide (Dorn & Kippenberger, 2008). This increased
immunogenicity is undesired since it may induce damage of the tissue to be treated, i.e. the skin (dermis
and/or epidermis).
The invention allows designing an oligonucleotide with acceptable RNA binding kinetics and/or
thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in
part determined by the melting temperature of an oligonucleotide (Tm; calculated with the
oligonucleotide properties calculator (www.unc.edu/~cail/biotool/oligo/index.html) for single stranded
RNA using the basic Tm and the nearest neighbor models), and/or the free energy of the AON-target
exon complex (using RNA structure version 4.5). If a Tm is too high, the oligonucleotide is expected to be
less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide, the
chemistry of the backbone (phosphodiester, phosphorothioate, phosphoramidate, peptide-nucleic acid,
etc.), the nature of the sugar moiety (ribose, deoxyribose, substituted ribose, intramolecular bridge) and
chemical modification of the nucleobase. Therefore, the range of Tm can vary widely.
In accordance with one aspect of the invention, new AONs are provided according to the invention by
microwalking the 5’ region of exon 73 with AONs. Thus, a novel 8 nucleotide motif (a putative ESE) has
been identified that forms a suitable target to design AONs according to the invention.
The length of the oligo selected by the present inventors was between 16 and 24 nucleotides, but a
different length is also possible. It is preferred to have a length that is long enough to allow for a stable
interaction with the target RNA and specificity for the target sequence but not longer than necessary, as
longer oligonucleotides are more expensive to manufacture and are more complex from an analytical
point of view. The 5' region of exon 73 may be probed for efficient exon skipping molecules, by making a
series of overlapping oligonucleotides that are tested in an in vitro assay for their efficacy of exon
skipping – as exemplified in the examples. The AONs that establish a satisfactory exon skipping efficacy
are then further selected on the basis of the manufacturability, immunogenicity and other usability
criteria provided herein.
The opposite strategy is also possible. In accordance with this strategy, the oligo’s are first designed
based on the manufacturability, immunogenicity and other usability criteria provided by the present
invention, and are then tested for exon skipping efficiency. A functional activity of said oligonucleotide is
preferably to induce the skipping of exon 73 (SEQ ID NO: 1) to a certain extent and/or at least
decreasing the production of an exon 73 containing mRNA, thereby increasing the production of a
shorter than wild-type yet functional collagen protein.
The exon skipping percentage or efficiency may be calculated by determining the concentration of wild-
type band amplified, divided by the concentration of the shortened (exon 73-free) band amplified, after
a given number of PCR cycles, times 100%, for any given primer set, provided the number of cycles is
such that the amplification is still in the exponential phase. Quantification can be performed using the
Bioanalyzer DNA1000 apparatus
Preferred AONs according to the invention are those showing a skipping percentage of more than 70%
in AON-treated cells compared to non-treated cells, more preferably more than 80%, still more
preferably more than 90%, as measured by RT-PCR analysis.
Preferably, an AON according to the invention, which comprises a sequence that is complementary to a
40 nucleotide sequence as shown in SEQ ID NO: 1 is such that the complementary part is at least 80%,
more preferably at least 90%, still more preferably at least 95%, most 100% complementary to the
target sequence. It is thus not absolutely required that all the bases in the region of complementarity
are capable of pairing with bases in the opposing strand. For instance, when designing the
oligonucleotide one may want to incorporate for instance a residue that does not base pair with the
base on the complementary strand. Mismatches may, to some extent, be allowed, if under the
circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridizing to the
complementary part. In this context, “sufficiently” means that the AONs according to the invention are
capable of inducing exon skipping of exon 73. Skipping the targeted exon may conveniently be assessed
by RT-PCR. The complementary regions are preferably designed such that, when combined, they are
specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of
complementary regions as this depends on the actual sequences in other (pre-)mRNA molecules in the
system. The risk that the oligonucleotide also will be able to hybridize to one or more other pre-mRNA
molecules decreases with increasing size of the oligonucleotide, while the length should not be too long
to create problems with manufacturability, purification and/or analytics.
It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain
the capacity to hybridize and/or bind to the targeted region(s) in the pre-mRNA, can be used in the
present invention. However, preferably at least the complementary parts do not comprise such
mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides
having such mismatches in one or more complementary regions. It is thought, that higher hybridization
strengths, (i.e. increasing number of interactions with the opposing strand) are favorable in increasing
the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the
complementarity is from 90% to 100%. In general this allows for 1 or 2 mismatch(es) in an
oligonucleotide of 20 nucleotides.
An exon skipping molecule of the invention is preferably an (antisense) oligonucleotide, which is
complementary to SEQ ID NO: 1.
Preferably, the length of the complementary part of the oligonucleotide is the same as the length of the
oligonucleotide, meaning there are no 5' or 3' ends of the oligo that do not form a base pair with the
target RNA. Thus a preferred length for an oligonucleotide of the invention is 24 nucleotides or less
e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
Particularly good results have been obtained with AONs having a length between 16 and 24 nucleotides.
An exon skipping molecule according to the invention may contain one of more DNA residues
(consequently a RNA “u” residue will be a “t” residue as DNA counterpart), or one or more RNA
residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein
below. SEQ ID NOs: 5-35 & 39-43 are RNA sequences, but the invention also encompasses each of these
sequences in DNA form, and also DNA/RNA hybrids of these sequences.
It is preferred that an exon skipping molecule of the invention comprises one or more residues that are
modified to increase nuclease resistance, and/or to increase the affinity of the antisense oligonucleotide
for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence
comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent
is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural
40 internucleoside linkage, or a combination of these modifications.
In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone.
Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane
backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones,
methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate,
sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide
backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that
have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged
backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the
phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are
resistant to enzymatic degradation and appear to function as antisense agents by arresting translation
or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides
have been successfully delivered to tissue culture cells by methods that physically disrupt the cell
membrane, and one study comparing several of these methods found that scrape loading was the most
efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids
are not effective mediators of morpholino oligonucleotide uptake in cells.
According to one embodiment of the invention the linkage between the residues in a backbone do not
include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or
more short chain heteroatomic or heterocyclic internucleoside linkages.
In accordance with this embodiment, a preferred nucleotide analogue or equivalent comprises a Peptide
Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-
1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The
backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein
the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone
comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem.
Commun, 495–497). Since the backbone of a PNA molecule contains no charged phosphate groups,
PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et
al. (1993) Nature 365, 566-568).
According to another embodiment of the invention, the backbone comprises a morpholino nucleotide
analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino
ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino
oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring,
and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic
phosphorodiamidate linkage.
In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a
substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly
destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide
analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate,
phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate
40 including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate,
phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate,
thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or
boranophosphate.
A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar
moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; -F; substituted or
unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may
be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; O-,
S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; -dimethylaminooxyethoxy; and
-dimethylaminoethoxyethoxy. The sugar moiety can be a furanose or derivative thereof, or a
deoxyfuranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or
derivative of. A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), in which the
2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. A preferred LNA comprises 2'-O,4'-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic
Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent
RNase H and nuclease resistant and increase the affinity for the target RNA.
It is understood by a skilled person that it is not necessary for all internucleosidic linkages in an
antisense oligonucleotide to be modified. For example, some internucleosidic linkages may be
unmodified, whereas other internucleosidic linkages are modified. AONs comprising a backbone
consisting of one form of (modified) internucleosidic linkages, multiple forms of (modified)
internucleosidic linkages, uniformly or non-uniformly distributed along the length of the AON are all
encompassed by the present invention. In addition, any modality of backbone modification (uniform,
non-uniform, mono-form or pluriform and all permutations thereof) may be combined with any form or
of sugar or nucleoside modifications or analogues mentioned below.
An especially preferred backbone for the AONs according to the invention is a uniform (all)
phosphorothioate (PS) backbone.
In another embodiment, a nucleotide analogue or equivalent of the invention comprises one or more
base modifications or substitutions. Modified bases comprise synthetic and natural bases such as
inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -
alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.
It is understood by a skilled person that it is not necessary for all positions in an antisense
oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues
or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position
within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the
invention has at least two different types of analogues or equivalents.
According to another embodiment AONs according to the invention comprise a 2’-O (preferably lower)
alkyl phosphorothioate antisense oligonucleotide, such as 2'-O-methyl modified ribose (RNA), 2’-O-
methoxyethyl modified ribose, 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose, and/or
substituted derivatives of these modifications such as halogenated derivatives.
An effective and preferred antisense oligonucleotide format according to the invention comprises 2’-O-
methyl modified ribose moieties with a phosphorothioate backbone, preferably wherein substantially all
ribose moieties are 2’-O-methyl and substantially all internucleosidic linkages are phosphorothioate
linkages.
40 It will also be understood by a skilled person that different antisense oligonucleotides can be combined
for efficiently skipping of exon 73 of the COL7A1 gene. A combination of two antisense oligonucleotides
may be used in a method of the invention, such as two antisense oligonucleotides, three different
antisense oligonucleotides, four different antisense oligonucleotides, or five different antisense
oligonucleotides targeting the same or different regions of exon 73 (fig. 1), as long as at least one AON is
one according to the invention.
An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense
oligonucleotide in cells, preferably skin cells. Examples of such moieties are cholesterols, carbohydrates,
vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to
antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine,
oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an
antibody, or a single chain antigen binding domain such as a camelid single domain antigen-binding
domain.
An exon skipping molecule according to the invention may be a naked (gymnotic) antisense
oligonucleotide or in the form of a conjugate or expressed from a vector (vectored AON). The exon
skipping molecule may be administrated using suitable means known in the art. When the exon skipping
molecule is a vectored AON, it may for example be provided to an individual or a cell, tissue or organ of
said individual in the form of an expression vector wherein the expression vector encodes a transcript
comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ
or individual via a gene delivery vehicle, such as a viral vector. In a preferred embodiment, there is
provided a viral-based expression vector comprising an expression cassette or a transcription cassette
that drives expression or transcription of an exon skipping molecule as identified herein. Accordingly,
the present invention provides a viral vector expressing an exon skipping molecule according to the
invention when placed under conditions conducive to expression of the exon skipping molecule. A cell
can be provided with an exon skipping molecule capable of interfering with sequences essential for, or
at least conducive to, exon 73 inclusion, such that such interference prevents, or at least reduces, exon
73 inclusion into the COL7A1 mRNA, for example by plasmid-derived antisense oligonucleotide
expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors.
Expression may be driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A
preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral
vector such as a lentivirus vector and the like. Also, plasmids, artificial chromosomes, plasmids usable
for targeted homologous recombination and integration in the mammalian (preferably human) genome
of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the
current invention are those vectors wherein transcription is driven from PolIII promoters, and/or
wherein transcripts are in the form of fusions with U1 or U7 transcripts, which yield good results for
delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are
PolIII driven transcripts. Preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such
fusions may be generated as described in the art (e.g.vide: Gorman L et al., 1998 or Suter D et al., 1999).
One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-
based vector. Single chain and double chain AAV-based vectors have been developed that can be used
for prolonged expression of antisense nucleotide sequences for highly efficient skipping of COL7A1 exon
40 73.
A preferred AAV-based vector for instance comprises an expression cassette that is driven by a
polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA
promoter.
The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven
expression cassette for expression of an antisense oligonucleotide of the invention for inducing skipping
of COL7A1 exon 73.
An AAV vector according to the present invention is a recombinant AAV vector and refers to an AAV
vector comprising part of an AAV genome comprising an encoded exon skipping molecule according to
the invention encapsidated in a protein shell of capsid protein derived from an AAV serotype as
depicted elsewhere herein. Part of an AAV genome may contain the inverted terminal repeats (ITR)
derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9
and others. Protein shell comprised of capsid protein may be derived from an AAV serotype such as
AAV1, 2, 3, 4, 5, 8, 9 and others. A protein shell may also be named a capsid protein shell. AAV vector
may have one or preferably all wild type AAV genes deleted, but may still comprise functional ITR
nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and
packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%,
90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in
insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this
context, functionality refers to the ability to direct packaging of the genome into the capsid shell and
then allow for expression in the host cell to be infected or target cell. In the context of the present
invention a capsid protein shell may be of a different serotype than the AAV vector genome ITR. An AAV
vector according to present the invention may thus be composed of a capsid protein shell, i.e. the
icosahedral capsid, which comprises capsid proteins (VP1, VP2, and/or VP3) of one AAV serotype, e.g.
AAV serotype 2, whereas the ITRs sequences contained in that AAV5 vector may be any of the AAV
serotypes described above, including an AAV2 vector. An “AAV2 vector” thus comprises a capsid protein
shell of AAV serotype 2, while e.g. an “AAV5 vector” comprises a capsid protein shell of AAV serotype 5,
whereby either may encapsidate any AAV vector genome ITR according to the invention.
Preferably, a recombinant AAV vector according to the present invention comprises a capsid protein
shell of AAV serotype 2, 5, 8 or AAV serotype 9 wherein the AAV genome or ITRs present in said AAV
vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9; such AAV vector is referred to as an
AAV2/2, AAV 2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV 5/9, AAV8/2, AAV 8/5, AAV8/8,
AAV8/9, AAV9/2, AAV9/5, AAV9/8, or an AAV9/9 vector, respectively.
More preferably, a recombinant AAV vector according to the present invention has tropism for dermal
and epidermal cells and comprises a capsid protein shell of AAV serotype 5 or 8. The AAV genome or
ITRs present in said vector may be derived from the same or a different serotype, such as AAV serotype
2; such vector is referred to as an AAV 2/5 or AAV 2/8 vector. AAV with a serotype 5 capsid have tropism
for dermal and epidermal cells, such as basilar and suprabasilar keratinocytes and dermal fibroblasts.
AAV vectors with a type 5 capsid display much higher transduction efficiencies compared to AAV with a
type 2 capsid (Keswani et al. Wound Repair Regen. 2012 ; 20(4): 592–600). Similarly, AAV with a capsid
of serotype 8 show tropism towards dermal fibroblasts and (mainly) suprabasilar keratinocytes.
Moreover, AAV 2/8 tend to be more efficient in transducing mammalian, preferably human dermal and
epidermal cells than AAV 2/5. However, transduction efficiency appears to depend on the timing of
40 administration during wound healing, AAV 2/2 showing higher transduction efficiencies than AAV 2/5
and AAV 2/8 at later time points (Keswani et al., supra).
Hence, AAV 2/2, AAV x/5 and AAV x/8 are preferred AAV to deliver AONs according to the invention and
their choice may be determined taking into account the time of administration and the cell types to be
targeted. These details can be readily worked out a person skilled in the art, in pre-clinical or clinical
studies.
A nucleic acid molecule encoding an exon skipping molecule according to the present invention
represented by a nucleic acid sequence of choice is preferably inserted between the AAV genome or ITR
sequences as identified above, for example an expression construct comprising an expression regulatory
element operably linked to a coding sequence and a 3’ termination sequence.
“AAV helper functions” generally refers to the corresponding AAV functions required for AAV replication
and packaging supplied to the AAV vector in trans. AAV helper functions complement the AAV functions
which are missing in the AAV vector, but they lack AAV ITRs (which are provided by the AAV vector
genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and
the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are
well known in the art, see e.g. Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or US 5,139,941,
incorporated herein by reference. The AAV helper functions can be supplied on a AAV helper construct,
which may be a plasmid. Introduction of the helper construct into the host cell can occur e.g. by
transformation, transfection, or transduction prior to or concurrently with the introduction of the AAV
genome present in the AAV vector as identified herein. The AAV helper constructs of the invention may
thus be chosen such that they produce the desired combination of serotypes for the AAV vector’s capsid
protein shell on the one hand and for the AAV genome present in said AAV vector replication and
packaging on the other hand.
“AAV helper virus” provides additional functions required for AAV replication and packaging. Suitable
AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and
vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the
host cell via vectors, as described in US 6,531,456 incorporated herein by reference.
Preferably, an AAV genome as present in a recombinant AAV vector according to the present invention
does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap
(capsid) genes of AAV. An AAV genome may further comprise a marker or reporter gene, such as a gene
for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a
chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known
in the art.
A preferred AAV vector according to the present invention is an AAV vector, preferably an AAV2/5,
AAV2/8, AAV2/9 or AAV2/2 vector, expressing an exon skipping molecule according to the present
invention comprising an antisense oligonucleotide, wherein said antisense oligonucleotide comprises or
consists of a sequence selected from the group consisting of: AON1, AON2, AON3, AON4, AON20,
AON21, AON22, AON23, AON24, AON24.1, AON24.2, AON24.3, AON24.3, AON.24.4, AON.24.5, AON25
and mh-AON1 as disclosed in Table 1 above.
Improvements in means for providing an individual or a cell, tissue, organ of said individual with an exon
skipping molecule according to the invention, are anticipated considering the progress that has already
thus far been achieved. Such future improvements may of course be incorporated to achieve the
mentioned effect on restructuring of mRNA using a method of the invention. An exon skipping molecule
40 according to the invention can be delivered as is to an individual, a cell, tissue or organ of said individual.
When administering an exon skipping molecule according to the invention, it is preferred that the
molecule is dissolved in a solution that is compatible with the delivery method.
Gymnotic AONs are readily taken up by most cells in vivo, and usually dissolving the AONs according to
the invention in an isotonic (saline) solution will be sufficient to reach the target cells, such as skin
(dermis and epidermis) cells. Alternatively, gymnotic AONs of the invention may be formulated using
pharmaceutically acceptable excipients, additives, stabilizers, solvents, colorants and the like. In
addition, or alternatively, gymnotic AONs may be formulated with any of the transfection aids
mentioned below.
Skin (dermis and epidermis) cells can be provided with a plasmid for antisense oligonucleotide
expression by providing the plasmid in an aqueous solution, such as an isotonic (saline) solution.
Alternatively, a plasmid can be provided by transfection using known transfection agents.
For intravenous, subcutaneous, intramuscular, intrathecal and/or intradermal administration it is
preferred that the solution is an isotonic (saline) solution. Particularly preferred in the invention is the
use of an excipient or transfection agents that will aid in delivery of each of the constituents as defined
herein to a cell and/or into a cell, preferably a skin (dermis and epidermis) cell. Preferred are excipients
or transfection agents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes
that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a
cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agents
comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or
derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine
copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), lipofectin™, DOTAP and/or viral
capsid proteins that are capable of self-assembly into particles that can deliver each constituent as
defined herein to a cell, preferably a skin (dermis or epidermis) cell. Such excipients have been shown to
efficiently deliver an oligonucleotide such as antisense nucleic acids to a wide variety of cultured cells,
including skin (dermis and epidermis) cells. Their high transfection potential is combined with an
acceptably low to moderate toxicity in terms of overall cell survival. The ease of structural modification
can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer
characteristics and toxicity.
Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components,
a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP
which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The
neutral component mediates the intracellular release. Another group of delivery systems are polymeric
nanoparticles.
Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA
transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to
formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an
oligonucleotide, across cell membranes into cells.
In addition to these common nanoparticle materials, the cationic peptide protamine offers an
alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system
can form so called proticles, which can be prepared by a simple self-assembly process to package and
mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the
40 above or other commercially available alternative excipients and delivery systems to package and
deliver an exon skipping molecule for use in the current invention to deliver it for the prevention,
treatment or delay of a disease or condition associated with a mutated exon 73 in the COL7A1 gene.
An exon skipping molecule according to the invention could be covalently or non-covalently linked to a
targeting ligand specifically designed to facilitate the uptake into the cell (especially a skin (dermis) cell),
cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to
peptide(-like) structures) recognizing cell, tissue or organ specific elements facilitating cellular uptake
and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release
of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
Therefore, in a preferred embodiment, an exon skipping molecule according to the invention is
formulated in a composition or a medicament or a composition, which is provided with at least an
excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or
enhancing its intracellular delivery.
Preferred delivery is through topical administration. As outlined in the accompanying examples such
may be through the use of a pharmaceutically acceptable hydrogel, such as Flaminal hydro™, which is a
hydrogel already used in patient care, (2) a hypromellose hydrogel or (3) a carbomer hydrogel. Topical
formulations that may be used for the topical delivery of the oligonucleotides of the present invention
are:
- Creams, either formulated as a water-in-oil or as an oil-in-water emulsion; the latter are more
cosmetically and aesthetically acceptable. Examples are Softisan based creams or cetomacrogol
creams.
- Gels: Solutions or suspensions, which contain a gelling agent that is uniformly distributed
throughout the liquid phase. Examples are hydrogels including , but not limited to hypromellose,
carbomer and alginate.
- Ointments. These usually contain <20% water and >50% hydrocarbons, waxes or polyols as the
vehicle. They have a more greasy skin feel than creams.
- Pastes: These contain a high percentage of finely dispersed solids with a stiff consistency.
- Suspensions, which are liquid preparations that contain solid particles dispensed in a liquid
vehicle. Some can be labeled as lotions.
- Lotions. These are fluid, somewhat viscous (emulsion) formulations, which share many
characteristics with suspensions, low viscosity gels and solutions.
- Foams, which are emulsions that have a fluffy consistency, when dispensed.
- Sprays, which are fine, small droplets of liquid, generated by a nozzle.
- Solutions, which are liquid products that are usually aqueous, but may contain other solvents
such as alcohols.
It is to be understood that if a composition comprises an additional constituent such as an adjunct
compound defined herein, each constituent of the composition may be formulated in one single
combination or composition or preparation. Depending on their identity, the skilled person will know
which type of formulation is the most appropriate for each constituent as defined herein. According to
one embodiment, the invention provides a composition or a preparation which is in the form of a kit of
parts comprising an exon skipping molecule according to the invention and a further adjunct compound
as defined herein.
40 If required, an exon skipping molecule according to the invention or a vector, preferably a viral vector,
expressing an exon skipping molecule according to the invention can be incorporated into a
pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.
Accordingly, the invention also provides a composition, preferably a pharmaceutical composition,
comprising an exon skipping molecule according to the invention, such as gymnotic AON or a viral vector
according to the invention and a pharmaceutically acceptable excipient. Such composition may
comprise a single exon skipping molecule according to the invention, but may also comprise multiple,
distinct exon skipping molecules according to the invention. Such a pharmaceutical composition may
comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant,
solubilizer and/or diluent. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant,
solubilizer and/or diluent may for instance be found in Remington, 2000. Each feature of said
composition has earlier been defined herein.
If multiple distinct exon skipping molecules according to the invention are used, concentration or dose
defined herein may refer to the total concentration or dose of all oligonucleotides used or the
concentration or dose of each exon skipping molecule used or added. Therefore in one embodiment,
there is provided a composition wherein each or the total amount of exon skipping molecules according
to the invention used is dosed in an amount ranged from 0.0001 and 100 mg/kg, preferably from 0.001
and 50 mg/kg, still more preferably between 0.01 and 20 mg/kg.
A preferred exon skipping molecule according to the invention, is for the treatment of DEB, or, more
generally, a mutated COL7A1 exon 73 related disease or condition of an individual. In all embodiments
of the present invention, the term “treatment” is understood to include the prevention and/or delay of
the disease or condition. An individual, which may be treated using an exon skipping molecule according
to the invention may already have been diagnosed as having DEB or a COL7A1 exon 73 related disease
or condition. Alternatively, an individual which may be treated using an exon skipping molecule
according to the invention may not have yet been diagnosed, but may be an individual having an
increased risk of developing DEB, or a COL7A1 exon 73 related disease or condition in the future given
his or her genetic background. A preferred individual is a human being. In a preferred embodiment the
mutated COL7A1 exon 73 related disease or condition is DEB.
The present invention further provides an exon skipping molecule according to the invention, such as an
AON, or a vector encoding an AON, such as a viral vector, according to the invention, or a composition
comprising an AON, or a vector encoding an AON, according to the invention for use as a medicine
e.g. for use in treating DEB or, more generally, a mutated COL7A1 exon 73 related disease or condition
of an individual (as discussed above).
The invention further provides the use of an exon skipping molecule according to the invention, such as
an AON, or a vector encoding an AON, such as a viral vector, according to the invention, or a
composition comprising an AON, or a vector encoding an AON, according to the invention in the
manufacture of a medicament for treating DEB or, more generally, a mutated COL7A1 exon 73 related
disease or condition of an individual (as discussed above).
The invention further provides a method for treating a mammal (preferably a human) carrying in its
genome a mutation in exon 73 of the COL7A1 gene causing a disease or disorder, including DEB,
comprising administering to the mammal (human) an AON, a (viral) vector, or a pharmaceutical
composition of the invention. These patients may suffer, or be at risk of developing DEB or a related
40 disorder. Related disorder, disease or condition also encompasses for example skin cancer (squamous
cell carcinoma), or other carcinomas, that may arise as a consequence of a collagen VII deficiency or
abnormality in the skin, or other organs of an individual, caused by or associated with a mutation in
exon 73 of the COL7A1 gene.
Further embodiments of the invention are AONs, viral vectors encoding AONs, and pharmaceutical
compositions comprising AONs according to the invention for use as a medicine to treat a mammal
(preferably a human) carrying in its genome a mutation in exon 73 of the COL7A1 gene.
Exon skipping molecules according to the invention may be administered to a patient systemically,
locally, topically, through administration that is orally, intraocularly, intrapulmonary, intranasally,
intramuscularly, subcutaneously, intradermally, rectally, by swallowing, injecting, inhalation, infusion,
spraying, in the form of (aqueous) solutions, suspensions, (oil-in-water) emulsions, ointments, lozenges,
pills etcetera.
Dosing may be daily, weekly, monthly, quarterly, once per year, depending on the route of
administration and the need of the patient.
Because of the early onset of disease, patients having or at risk of developing a disease, disorder or
condition caused by or associated with a mutated exon 73 of the COL7A1 gene, including DEB, may be
treated in utero, directly after birth, from 1, 2, 3, 6 months of age, from one year of age, from 3 years of
age, from 5 years of age, prior to or after the onset of symptoms, to alleviate, retard development, stop
or reverse the symptoms of disease and the like.
A treatment in a use or in a method according to the invention is at least one week, at least one month,
at least several months, at least one year, at least 2, 3, 4, 5, 6 years or chronically, even during a
patient’s entire life. Each exon skipping molecule or exon skipping oligonucleotide or equivalent thereof
as defined herein for use according to the invention may be suitable for direct administration to a cell,
tissue and/or an organ in vivo of individuals already affected or at risk of developing a mutated COL7A1
exon 73 related disorder, disease or condition, and may be administered directly in vivo, ex vivo or in
vitro. The frequency of administration of an oligonucleotide, composition, compound or adjunct
compound of the invention may depend on several parameters such as the age of the patient, the
nature of the exon skipping molecule (e.g. gymnotic AON or vectored AON, such as AAV or lentiviral
vector expressed AONs), the dose, the formulation of said molecule and the like.
Dose ranges of an exon skipping molecule, preferably an oligonucleotide according to the invention are
preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous
protocol requirements exist. An oligonucleotide as defined herein may be used at a dose range from
0.0001 to 100 mg/kg, preferably from 0.01 to 20 mg/kg. The dose and treatment regime may vary
widely, depending on many factors, including but not limited to the route of administration (e.g.
systemic versus topically), whether the oligo is administered as a gymnotic AON or as vectored AON, the
dosing regimen, the age and weight of the patient, and so forth.
In a preferred embodiment, a viral vector, preferably an AAV vector as described earlier herein, as
delivery vehicle for a molecule according to the invention, is administered in a dose ranging from 1x10
17 10 14 10
– 1x10 virus particles per injection, more preferably from 1x10 – 1x10 , and most preferably 1x10 –
1x10 virus particles per injection.
It will be clear to a person having ordinary skill in the art to which this invention pertains, that the details
of treatment will need to be established in accordance with and depending on such factors as the
sequence and chemistry of the oligonucleotide(s), the route of administration, the formulation, the
40 dose, the dosing regimen, the format (viral vector or gymnotic oligonucleotide), the age and weight of
the patient, the stage of the disease and so forth, which may require further non-clinical and clinical
investigation.
The invention further provides a method for preventing, or at least reducing, COL7A1 exon 73 inclusion
in a cell comprising contacting the cell, preferably a skin cell (dermal fibroblast), with an exon skipping
molecule according to the invention, such as a gymnotic AON or a (viral) vector encoding an AON
according to the invention, or a composition according to the invention. The features of this aspect are
preferably those defined earlier herein.
Unless otherwise indicated each embodiment as described herein may be combined with another
embodiment as described herein.
The ability of an exon skipping molecule, such as an AON according to the invention, or a (viral) vector
encoding such AON, to prevent, or at least reduce, mutated COL7A1 exon 73 inclusion, when the
COL7A1 gene is expressed in a mammalian (preferably human) cell, and to bind to the mammalian
(human) COL7A1 pre-mRNA under physiological conditions in a region affecting selection of the 5’ splice
acceptor, and thereby reduce inclusion of the mutated exon 73 into the COL7A1 mRNA, can be
conveniently assessed using the assays disclosed in the experimental section herein. In particular, the
exon skipping molecule can be incubated with a cell containing exon 73 (not necessarily mutated) of the
COL7A1 gene to assess its ability to reduce production by the cell of mRNA which includes exon 73,
e.g. by RT-PCR (which can be quantified using a Bioanalyzer apparatus), as described herein in the
experimental section and the examples.
As can be observed in the experimental section and the Examples herein, at the RNA level, addition of
various AONs according to the invention targeting exon 73 of the COL7A1 gene indeed resulted in a
mRNA lacking exon73, leading to the production of a shorter but functional collagen VII protein.
In fibroblasts (that can be derived from the dermis part of the skin), collagen VII is abundantly
expressed. Therefore, it is to be expected that addition of AONs to cultured fibroblasts from DEB
patients will result in an increased amount of shortened but functional collagen VII protein that is
detectable on Western blot, and as such will demonstrate that AON-based therapy will not only redirect
splicing of the COL7A1 mRNA but will also result in restoring collagen VII functionality.
The terms “adenine”, “guanine”, “cytosine”, “thymine”, “uracil” and hypoxanthine (the nucleobase in
inosine) refer to the nucleobases as such.
The terms adenosine, guanosine, cytidine, thymidine, uridine and inosine, refer to the nucleobases
linked to the (desoxy)ribosyl sugar.
The term “nucleoside” refers to the nucleobase linked to the (deoxy)ribosyl sugar.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting
sense to mean that items following the word are included, but items not specifically mentioned are not
excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the
possibility that more than one of the element is present, unless the context clearly requires that there
be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
The word “include” and all of its tenses and conjugations, is to be read as “include, but is not limited to”.
The word “exon skipping molecule” is meant to include gymnotic AONs and vectored AONs, including
viral vectors, capable of expressing AONs in a compatible cell.
The word “about” or “approximately” when used in association with a numerical value (e.g. about 10)
preferably means that the value may be the given value (of 10) plus or minus 5% of the value.
The sequence information as provided herein should not be so narrowly construed as to require
inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously
identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of
the polypeptide obtainable by expression of the gene present in SEQ ID NO: 1 containing the nucleic
acid sequence coding for the polypeptide should prevail.
EXAMPLES
Example 1: mRNA analysis of exon 73
To detect the presence of mRNA of exon 73 in mRNA of COL7A1 extracted mRNA of both HeLa cells and
human primary fibroblasts (HPF) were used. Culturing of cells was performed in (a) Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for HeLa, or (b) DMEM AQE
supplemented with 20% FBS and 1% natrium pyruvate for HPF cells. All cells were grown at 37°C 5% C0 .
To determine the exon skip efficiency of described AONs, cells were seeded at 60.000 cells/well (HeLa)
into 12-well plates or 150.000 cells/well (HPF) into 6-well plates. After 24 hours of allowing cells growth
cells were transfected with 100nm AON-maxPei complex. RNA isolation was performed with the
ReliaPrep™ RNA Cell Miniprep System (Promega), subsequently cDNA was made using the Thermo
Scientific Verso kit. PCR for exon 73 was performed with FW primer (5’-GCTGGCATCAAGGCATCT-3’;
SEQ ID NO: 51) located at the exon 71-72 boundary and RV primer (5’-TCCTTTCTCTCCCCGTTCTC-3’;
SEQ ID NO: 52) located within exon 74. PCR products were visualized with the Bioanalyzer using
DNA1000 chips and software Expert 2100 was used for product length analysis.
Skipping efficiencies are shown in Table 2, and Figure 3 shows lab-on-a-chip results. The AONs according
to the invention designated AON1 to AON4, AON20 to AON25 (including AONs 24.1 to 24.5) and m-h
AON1 have the best efficiency, with >70% of mRNA having exon 73 removed. The effective AONs target
the 5' end of the pre-mRNA.
Table 2: Efficiency of exon 73 exclusion from mRNA in HPF and HeLa cells
AON sequence 5’ – 3’ SEQ ID Notes
HPF HeLa NO
82% 96% 37
UCUCCACGGUCGCCCUUCAGCCCGCGUUCU
ESE73.3
80% 73% UCUCCACGGUCGCCCUUCAGCCCGC 38
ESE73.7
67% 86% 5
UCUCCAGGAAAGCCGAUGGGGCCC
AON1
69% 85% AGCCCGCGUUCUCCAGGAAAGCCGA 6
AON2
67% 92% 7
GUCGCCCUUCAGCCCGCGUUCUCCA
AON3
91% 83% ACGGUCGCCCUUCAGCCCGCGUU 8
AON4
% 3% CCCCUGAGGGCCAGGGUCUCCACGG 9
AON5
2% 0% CAGACCAGGUGGCCCCUGAGGGCCA 10
AON6
CCAAGGGCCAGACCAGGUGGCCCC 11
AON7 4% 0%
CCAGACCAGGUGGCCCCUGAGGGCC 12
AON8 0% 0%
UCUCCCCAAGGGCCAGACCAGG 13
AON9 0% 0%
GGAAGGCCCGGGGGGGCCCCUCUC
AON10 0% 0%
CCGGCAAGGCCGGAAGGCCCGGGG 15
AON11 6% 6%
AGGCUUUCCAGGCUCCCCGGCAAG 16
AON12 0% 0%
CGGGAAUACCAGGCUUUCCAGGCU
AON13 0% 2%
UGCCUGGGAGCCCGGGAAUACCA 18
AON14 17% 25%
CCCACACCCCCAGCCCUGCCUGGG
AON15 8% 8%
CCUCUCCCACACCCCCAGCCCU 20
AON16 8% 0%
UCUCUCCUGGCCUUCCUGCCUCU 21
AON17 9% 9%
CACCCUCUCUCCUGGCCUUCCU
AON18 11% 13%
CCAGCCUCACCCUCUCUCCUGG 23
AON19 0% 7%
CUCCAGGAAAGCCGAUGGGGCCC
24 AON1-1N at 3”
AON20 74% 100%
UCCAGGAAAGCCGAUGGGGCCC 25
AON21 58% 89% AON1-2N at 3”
CCAGGAAAGCCGAUGGGGCCC 26
AON22 64% 85% AON1-3N at 3”
CUCCAGGAAAUCCGAUGGGGCCcu
27 AON1-N at 3'+ 1 at 5'
AON23 64% 83%
UCCAGGAAAGCCGAUGGGGCCcug 28 AON1-2N at 3'+2 at 5'
AON24 72% 93%
AON24.1 32% 73% UCCAGGAAAGCCGAUGGG
UCCAGGAAAGCCGAUGG 40
AON24.2 50% 88%
UCCAGGAAAGCCGAUG 41
AON24.3 49% 79%
CUCCAGGAAAGCCGAUGG
AON24.4 53% 86%
UCUCCAGGAAAGCCGAUG 43
AON24.5 66% 89%
CCAGGAAAGCCGAUGGGGCCcugc
29 AON1-3N at 3'+3 at 5'
AON25 54% 92%
AGGAAAGCCGAUGGGGCCcugcag 30
AON26 22% 49% 2N shift towards 5'
GAAAGCCGAUGGGGCCcugcagga 31
AON27 40% 37% 4N shift towards 5'
AAGCCGAUGGGGCCcugcaggagu
32 6N shift towards 5'
AON28 20% 47%
GCCGAUGGGGCCcugcaggagugg 33
AON29 5% 0% 8N shift towards 5'
GAUGGGGCCcugcaggaguggaa
34 11N shift towards 5'
AON30 6% 7%
UCCAGGAAAG 44
AON31 0% 0%
CGUUCUCCAGGAAAGCCGAUG 35
76% 91%
m-hAON 1
CCUGAGGGCCAGGGUCUCCACG
m-hAON 2 0% 16%
Two of the AONs that showed satisfactory exon skipping efficiency were truncated by removing a
varying number of nucleotides at the 3’ end in order to avoid the occurrence undesirable G-tetrads.
These AONs are shown in Table 3.
Table 3: Truncated versions of AON24 and AON31
Name AONs sequence SEQ ID RNA binding sequence SEQ ID length
NO NO
UCCAGGAAAGCCGAUGGG CCCAUCGGCUUUCCUGGA
AON 24.1 39 45 18
UCCAGGAAAGCCGAUGG CCAUCGGCUUUCCUGGA
24.2 40 46 17
UCCAGGAAAGCCGAUG CAUCGGCUUUCCUGGA
24.3 41 47 16
CCAUCGGCUUUCCUGGAG
CUCCAGGAAAGCCGAUGG
24.4 42 48 18
UCUCCAGGAAAGCCGAUG CAUCGGCUUUCCUGGAGA
24.5 43 49 18
CUUUCCUGGA
UCCAGGAAAG
AON 31 44 50 10
These AONs efficiently reduced exon 73 inclusion into the COL7A1 mRNA (see Table 1), while being
devoid of any sequences that are less desirable from a manufacturability, purification and analytical
perspective, or the chance of overall loss of function due to multiplexing.
The functionality of Collagen VII without the exon 73 can be addressed using several in vitro methods
described in literature:
1. Protein analysis, both size and correct assembly of the α1-collagen chains, using western
blotting (Titeux et al 2010). Of note, due to the small size of the skipped exon and the large
size of the wild type protein, the apparent difference in protein size may not be picked-up.
2. Thermal stability analysis of the collagen VII homotrimer, by using western blotting under
non-reduced conditions. Wild-type collagen VII is comprised of three α1-collagen a chains,
and has a Tm of 41°C (Mecklenbeck et al., 2002).
3. Cell migration analysis using colloidal gold or scratch assay. Compare the motility of
fibroblasts and/or keratinocytes that express wild-type collagen VII vs the truncated protein
without exon 73 (Chen et al. 2002).
4. Cell adhesion to various extracellular matrix components can be assessed, e.g. to collagen
IV, laminin-332, laminin-1 or fibronectin (Chen et al. 2002).
The inventors postulate that the AONs shown to perform the best in terms of preventing, or at least
reducing, exon 73 inclusion into the mammalian (preferably human) COL7A1 mRNA will provide
satisfactory results in terms of collagen VII functionality, as can be readily assessed using the above
methods from the prior art. Moreover, the AONs that comprise no more than two (preferably no more
than one, such as one,) CpG will perform satisfactorily in terms of in vivo immunogenicity. Hence, the
most preferred AONs of the invention are candidates for development into therapeutics, suitable for
therapy in humans suffering from, or at risk of suffering from, forms of dystrophic epidermolysis bullosa
associated with mutations in exon 73 of the COL7A1 gene.
Example 2: Topical delivery of mh-AON1 using an ex vivo porcine skin model
Current wound management for DEB patients is mainly focused on wound care, management of itching
and pain and early diagnosis of squamous cell carcinoma. Wound care includes cleaning and sterilizing
of the wounds by the means of (chloride) baths, the use of chlorhexidine as a disinfectant and other
antimicrobial creams. In addition the wounds are hydrated and moisturized using hydrogels to reduce
pain and itch. Finally, wound care involves bandaging with different types of dressings/silicone foams to
protect and reduce friction to the skin, prevent contamination, prevent sticking of material, absorb
liquid from the wounds, to prevent blisters from growing in size, the blisters are punctured and drained
to decrease the pressure from within.
Topical delivery of mh-AON1 provides a couple of advantages, firstly due to the local delivery there will
be direct delivery to the target cells, keratinocytes and fibroblasts. Secondly due to the local
administration systemic absorption will only be minor, resulting in less systemic toxicity (Wraight &
White Pharmacol Ther 2001 Apr;90(1):89-104). Finally, it has been shown that after topical
administration of oligonucleotides local concentrations in the dermis and epidermis can be up to 150
(for the dermis) and 4000 (for the epidermis) times as high as after systemic administration (Metha et
al., J Invest Dermatol. 2000 Nov;115(5):805-12).
To investigate the topical delivery of mh-AON1 an in-house ex vivo porcine skin model was established.
Porcine skin is considered to be highly similar to human skin, with equal epidermal thickness and barrier
40 properties of the stratum corneum. For the delivery studies, porcine ex vivo skin was received, cut to a
thickness between 0.8 and 1.4 mm and cultured at the air-liquid interface with the apical site air
exposed. In the wounds of DEB patients the epidermis is completely separated from the dermis,
therefore these wounds were mimicked by mechanically removing the epidermis completely. To assess
the skin penetration of mh-AON1 into intact or blister-like ex vivo porcine skin, the oligonucleotide was
either formulated into PBS or into a hydrogel, part of DEB standard wound care. After exposure to mh-
AON1 the skin pieces were fixed in 4% formalin, processed and embedded in paraffin for histological
assessment using hematoxylin as a counterstaining for morphology. Since the oligonucleotide was
conjugated to a Cy5 label the site of mh-AON1 could be visualized by fluorescent microscopy.
mh-AON1 formulated in PBS
Intact and blister-like ex vivo porcine skin pieces were incubated with 25µg of mh-AON1 formulated into
PBS for 24 hours after which they were processed for analysis. Results show that mh-AON1 added onto
intact porcine skin pieces will not penetrate the stratum corneum (Figure 4a-b). However, when the mh-
AON1 formulation is incubated on the blister-like porcine skin, it was observed that the oligonucleotide
had penetrated into the dermis (figure 4c-f).
mh-AON1 formulated into hydrogels
For application onto patient wounds it is beneficial to incorporate mh-AON1 into an ointment or gel.
Since DEB patients use hydrogels as part of their wound care, e.g. to moisturize the wounds and thereby
decrease pain and itch, it was tested whether mh-AON1 could be incorporated into a hydrogel as well.
For this purpose three different hydrogels were used: (1) flaminal™, which is a hydrogel already used in
patient care, (2) a hypromellose hydrogel and (3) a carbomer hydrogel both formulated in-house. All
hydrogels are already commonly used in clinical settings. The hydrogel formulations were prepared with
and without oligonucleotide, and spread on the skin pieces, 25 µg mh-AON1 was formulated into 50mg
of gel for each skin piece, giving an end concentration of 0.5 mg/ml oligonucleotide.
It was observed that mh-AON1 formulated into either flaminal™, hypromellose or carbomer hydrogels
could never penetrate the intact stratum corneum of the ex-vivo porcine skin pieces (figure 5a, c, e, g).
However all three hydrogels could deliver the oligonucleotide into the dermis of the blister-like porcine
skin where the epidermis was removed (figure 5b, d, f, h). Optimization the hydrogels is ongoing and
selection of the final formulation will be based on the dermal penetration depth, local tolerability, pH of
mh-AON1 combination, stability of the mh-AON1 hydrogel formulation and the release from mh-AON1
from the hydrogel.
Conclusion
DEB patients suffer greatly from their fragile skin due to blisters, wounds and ulcerations. Moreover
they need constant wound care. mh-AON1 was therefore assessed via the topical route of delivery.
Blister-like skin was created by removing the epidermis, including stratum corneum which mimics the
DEB patient skin. It was demonstrated that mh-AON1 formulated in either PBS or a hydrogel is able to
penetrate blister-like skin and reaches the dermis. These results support that topical administration to
the patient’s skin wounds is a feasible approach to deliver mh-AON1 to the target cells in the skin.
Moreover, these findings support that a formulation resembling EB standard of care seems suitable in
delivery of mh-AON1.
Example 3: Efficacy testing at the mRNA level
Two different cell types were used to assess the efficacy of mh-AON1: (1) HeLa and (2) skin derived
40 human primary fibroblasts (HPF) from healthy individuals. Both cell types express COL7A1 mRNA and
produce the collagen type VII protein. mh-AON1 as disclosed herein has been designed to exclude exon
73 from the COL7A1 mRNA, and thus exclude mutations from the transcript. Since mh-AON1 targets the
splicing process, the most direct measurable outcome of efficacy is the profiling and quantification of
COL7A1 transcripts (wild type and Δ73) with and without the addition of mh-AON1.
Profiling and quantification of COL7A1 mRNA Level through Polymerase Chain Reaction (PCR)
PCR is a straightforward technology which enables the logarithmic amplification of a specific DNA
(cDNA) sequence. COL7A1 sequence-specific primers, flanking exon 73, were used to perform the PCR
reaction. Afterwards the products formed were visualized using lab on a chips technology that allows
discrimination of different fragment length products and the quantitative analysis based on yield.
For exon 73 skip experiments, HPF and HeLa cells were transfected with mh-AON1 at a concentration of
100 nM using polyethyleneimine (Poly I:C) as a transfection vehicle. 24 or 40 hours post transfection,
the cells were harvested, whole mRNA isolated, cDNA synthesized and a PCR performed using COL7A1
specific primers, one in exon 69 and one in exon 74. As a negative control a scrambled (SCRM) version of
the mh-AON1 oligonucleotide was taken along.
Results show that treatment with mh-AON1 leads to efficient exclusion of exon 73 from the COL7A1
mRNA compared to SCRM treated cells (Figure 6) as determined by PCR. Furthermore, the level of wild
type mRNA in untreated cells was comparable to the level of total COL7A1 mRNA in treated cells. Since
the PCR/bioanalyzer method is informative but not absolute quantitative, these initial findings were
followed up by using droplet digital PCR assays which offer highly accurate and absolute quantification
of nucleic acid fragments.
Profiling and quantification of COL7A1 mRNA transcripts with droplet digital PCR
Droplet digital PCR (ddPCR) provides a highly accurate and absolute quantification of nucleic acids
through the partition of the PCR sample into thousands of droplets. The COL7A1 mRNA/cDNA PCR input
was adjusted in such a way that each droplet contains either one or none COL7A1 cDNA molecule. To
allow detection of the template, a probe specific for wild type or Δ73 COL7A1, was added to the PCR
mix. The location of these probes are depicted in Figure 7. One of the probes is specific for the wild type
product, while the other probe is specific only for the Δ73 COL7A1 product. This probe upon binding to
the template gets hydrolyzed and become fluorescent, so that after PCR amplification is performed, the
fluorescent droplets containing the target sequence can be counted. Using Poisson statistical analysis of
the numbers of positive and negative droplets, absolute quantitation of wild type or Δ73 COL7A1 mRNA
molecules in the sample can be calculated.
HeLa cells were transfected with either 50, 100 or 200nM mh-AON1 to establish a dose-response profile
for mh-AON1. Results from 24 h after transfection show that treatment with mh-AON1 results in both
COL7A1 wild type transcripts and Δ exon 73 transcripts. These results corroborate the observations seen
with PCR. The dose of 50nM already gives almost maximum effect after 24 h. After 40 h a small increase
in the % of Δ exon 73 transcripts was observed for the 50nM and 200nM transfection (Figure 8).
Example 4: In vitro immunogenicity tests
Oligonucleotides have the potential to cause activation of pattern recognition receptors (PRR) of the
vertebrate innate immune system. The best studied family of PRR receptors are the toll-like receptors
(TLRs). TLRs are a class of proteins that play a key role in the innate immune system. They are single,
membrane-spanning, non-catalytic receptors that are usually expressed in macrophages and dendritic
40 cells that recognize structurally-conserved molecules derived from microbes. TLRs that are activated by
different types of nucleic acids are those located on endosomes: TLR 3 (recognizes double stranded
RNA); TLR7/8 (recognizes double and single stranded RNA); and TLR9 (recognizes CpG-DNA).
Upon recognition of these components by the PRRs, a specific ‘antimicrobial’ immune response is
triggered. TLR activation results in the activation of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB), Interferon regulatory factor 3 (IRF-3) and activator protein 1 (AP-1). Activation
of AP-1, IRF-3 and NF-κB results in the production of inflammatory cytokines, type-I interferons and
other mediators of the innate immune response. These processes not only trigger immediate host
defensive responses such as inflammation, but also prime and orchestrate antigen-specific adaptive
immune responses.
In vitro exposure of primary human peripheral blood mononuclear cells (PBMC) to mh-AON1 was used
to assess (systemic) drug-specific immune responses and immunotoxicity. The in vitro assay using PBMC
is an established preclinical test using the production of (inflammatory) cytokines as surrogate marker
for systemic immune responses. The PBMC assay enables prediction of tolerability as a factor of the
immunogenicity and allergenicity potential of investigational compounds, and could enable an
estimation of a safe dosing range for these compounds.
For the studies of mh-AON1, in-house isolated PBMC were used, acquired from buffy coats of healthy
blood bank donors. Production of the key pro-inflammatory cytokines in the culture supernatant was
assessed after 24 h of stimulation with mh-AON1 at concentrations ranging from 10 nM to 1 µM. In
addition, the Ramos-Blue (Invivogen, human B cells) reporter cell line with chromosomal integration of a
secreted embryonic alkaline phosphatase reporter construct inducible by NF-κB and/or AP-1 was used
to assess general PPR-mediated immune activation by mh-AON1 and AON73.24.5. Ramos-Blue cells
express the relevant set of TLRs, including: TLR3, -7/8 and -9. Activation NF-κB and/or AP-1 was
measured after 24 h of stimulation with mh-AON1 or AON73.25.4 at concentrations ranging from 10 nM
to 1 µM. Moreover, the viability of the PBMC and Ramos-Blue after treatment with mh-AON1 was
analyzed by measuring the fluorescent resorufin in the culture supernatant to assess potential cytoxic
effect of mh-AON1. Viable cells convert the non-fluorescent resazurin into fluorescent resorufin.
Results in human PBMC
Stimulation of human PBMC with the positive controls LPS (TLR4 agonist) and R848 (TLR7/8 agonist)
resulted in significantly increased concentrations of all measured cytokines, except IL-3, in the culture
supernatant. Moreover, stimulation with CpG DNA (TLR9 agonist) or Poly (I:C) (TLR3 agonist) induced a
similar pattern of cytokines, although to a lesser extent. A Heat map depicting the significance levels of
cytokine concentrations in culture supernatant after stimulation with mh-AON1 or the positive controls
compared to saline-treated human PBMC is shown in Figure 9a. Importantly, stimulation of human
PBMC with mh-AON1 concentrations ranging from 10 nM to 1 µM did not results in increased
concentrations of any of the measured cytokines in the culture supernatant, with the exception of IFN-
α2 at t he lowest concentration mh-AON1 (Figure 9a). However, since the increase in concentration of
IFN-α2 in the supernatant after stimulation with mh-AON1 is not dose dependent, this was considered
as an experimental outlier or technical error (Figure 9b). Finally, there were no signs of cytotoxicity 24 h
after treatment with mh-AON1 (Figure 9c). In contrast, there was a slight increase in viability observed
after treatment with R848, or 10 nM and 100 nM mh-AON1 suggesting enhanced cell survival, increased
cell metabolism or even increased proliferation/differentiation.
40 Results in Ramos-Blue cells
Results of the immunogenicity assay carried out in the human Ramos Blue cell line showed no activation
of NF-κB and/or AP-1 after 24 h treatment with mh-AON1 or AON73.24.5 at concentrations ranging
from 10 nM to 1 µM (Figure 10a). In contrast, the positive controls Poly(I:C) (1µg/ml), CpG (10µg/ml)
and R848 (1µM) did induce activation of NF-κB and/or AP-1. LPS had no effect, since TLR4 is not
expressed on Ramos-Blue. Moreover, there were no signs of cytotoxicity 24 h after treatment with MH-
AON1 (figure 10b) confirming the results obtained in human PBMC.
It will be understood that the invention is described above by way of example only and modifications
may be made whilst remaining within the scope and spirit of the invention.
Claims (18)
1. An antisense oligoribonucleotide capable of preventing or reducing exon 73 inclusion into the human COL7A1 mRNA, when said mRNA is produced by splicing from a pre-mRNA in a mammalian cell, characterized in that the oligoribonucleotide is capable of annealing to the (SRp40/SC35 binding / ESE) element in exon 73 characterized by the sequence 5’-UUUCCUGG-3’ (SEQ ID NO: 4), wherein the oligoribonucleotide has a length of between 16 and 24 nucleotides.
2. An antisense oligoribonucleotide according to claim 1, characterized in that it has no more than one CpG sequence.
3. An antisense oligoribonucleotide capable of preventing or reducing exon 73 inclusion into the human COL7A1 mRNA, when said mRNA is produced by splicing from a pre-mRNA in a mammalian cell, characterized in that the oligoribonucleotide is selected from the group consisting of the antisense oligoribonucleotides of SEQ ID NO: 5, 6, 7, 8, 24, 25, 26, 27, 28, 39, 40, 41, 42, 43, 29 and
4. An antisense oligoribonucleotide according to claim 3, characterized in that the oligoribonucleotide is the antisense oligoribonucleotide of SEQ ID NO: 35.
5. An antisense oligoribonucleotide according to any one of claims 1 to 4, characterized in that the internucleosidic linkages are chemically modified.
6. An antisense oligoribonucleotide according to claim 5, wherein the internucleosidic linkage modification is a phosphorothioate linkage.
7. An antisense oligoribonucleotide according to any one of claims 1 to 6, characterized in that the sugar moieties of the oligoribonucleotide are lower 2'-O-alkyl.
8. An antisense oligoribonucleotide according to claim 7, wherein the 2'-O-alkyl is a 2'-O-methyl substituted sugar moiety.
9. An antisense oligoribonucleotide according to any one of claims 1 to 8, characterized in that the length is selected from the group consisting of 16, 17 or 18 nucleotides.
10. An antisense oligoribonucleotide according to any one of claims 1 to 8, characterized in that the length is selected from the group consisting of 19, 20 or 21 nucleotides.
11. An antisense oligoribonucleotide according to any one of claims 1 to 8, characterized in that the length is selected from the group consisting of 22, 23 or 24 nucleotides.
12. An antisense oligoribonucleotide according to any one of claims 1 to 11, characterized in that the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 70%.
13. An antisense oligoribonucleotide according to any one of claims 1 to 11, characterized in that the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 80%.
14. An antisense oligoribonucleotide according to any one of claims 1 to 11, characterized in that the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 90%.
15. An antisense oligoribonucleotide according to any one of claims 1 to 11, characterized in that the oligoribonucleotide contains a modified base, wherein said modified base comprises a 2’-O-alkyl-O- alkyl modification.
16. An antisense oligoribonucleotide according to any one of claims 1 to 11, characterized in that the oligoribonucleotide contains a modified base, wherein said modified base comprises a 2’- methoxyethoxy modification.
17. Use of antisense oligoribonucleotide according to any one of claims 1 to 16 in the manufacture of a medicament for treating an individual having dystrophic epidermolysis bullosa.
18. A composition comprising an antisense oligoribonucleotide according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB1504124.7A GB201504124D0 (en) | 2015-03-11 | 2015-03-11 | Oligonucleotides |
GB1504124.7 | 2015-03-11 | ||
PCT/EP2016/055360 WO2016142538A1 (en) | 2015-03-11 | 2016-03-11 | Oligonucleotides matching col7a1 exon 73 for epidermolysis bullosa therapy |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ736083A NZ736083A (en) | 2021-08-27 |
NZ736083B2 true NZ736083B2 (en) | 2021-11-30 |
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