Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene.

Full text

Page 1

143
Annemieke Aartsma-Rus (ed.), Exon Skipping: Methods and Protocols, Methods in Molecular Biology, vol. 867,
DOI 10.1007/978-1-61779-767-5_10, © Springer Science+Business Media, LLC 2012
Chapter 10
Optimizing Antisense Oligonucleotides Using
Phosphorodiamidate Morpholino Oligomers
Linda J. Popplewell , Alberto Malerba , and George Dickson
Abstract
Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human
DMD gene. Selective removal of exons fl anking an out-of-frame DMD mutation can result in an in-frame
mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like func-
tionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be
designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to
correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional trun-
cated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD
patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this fi eld sug-
gest that it is likely that AO-induced exon skipping will be the fi rst gene therapy for DMD to reach the
clinic. However, it should be noted that personalized molecular medicine may be necessary, since the vari-
ous reading frame-disrupting mutations are spread across the DMD gene. The different deletions that
cause DMD would require skipping of different exons, which would require the optimization and clinical
trial workup of many specifi c AOs. This chapter describes the methodologies available for the optimization
of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specifi c
exons on the DMD gene.
Key words: Optimization , Exon skipping , DMD , Antisense oligonucleotides , Functional analysis ,
Phosphorodiamidate morpholino oligomers

Duchenne muscular dystrophy (DMD) is a disease caused by the
lack of functional dystrophin protein in skeletal muscles, as a result
of frame-disrupting deletions or duplications or, less commonly,
nonsense or missense mutations in the DMD gene ( 1 ) . Mutations
that maintain the reading frame of the gene and allow expression
1. Introduction

Page 2

144L.J. Popplewell et al.
of semifunctional but internally deleted dystrophins are generally
associated with the less severe Becker muscular dystrophy
(BMD) ( 1, 2 ) .
Transforming an out-of-frame DMD mutation into its in-
frame BMD counterpart with antisense oligonucleotides (AOs) is
the basis of the potentially exciting exon skipping therapy for DMD
( 3 ) (see Chapter 7 for a detailed review). The hybridization of AOs
to specifi c RNA sequence motifs prevents assembly of the spli-
ceosome so that it is unable to recognize the target exon(s) in the
pre-mRNA and include them in the mature gene transcript ( 4, 5 ) .
AOs have been used to induce skipping of specifi c exons such that
the reading frame is restored and truncated dystrophin expressed
in vitro in DMD patient cells ( 6– 9 ) , and in animal models of the
disease in vivo ( 10– 14 ) .
Initial proof-of-principle clinical trials, using two different AO
chemistries (phosphorothioate-linked 2 ¢ -O-methyl modifi ed bases
(2 ¢ OMePS) ( 15 ) and phosphorodiamidate morpholino oligomer
(PMO) ( 16 ) ) for the targeted skipping of exon 51 of the DMD
gene after intramuscular injection, have been performed recently
with encouraging results. While both chemistries (see Chapter 23
for a review on oligo chemistries) have excellent safety profi les
( 17, 18 ) , PMOs appear to produce more consistent and sustained
exon skipping in the mdx mouse model of DMD ( 19– 21 ) , human
muscle explants ( 22 ) , and dystrophic canine muscle cells in vitro
( 23 ) . However, for some human exons, 2 ¢ OMePS and PMO AOs
performed equally well ( 18 ) . Since the mutations that cause DMD
are so diverse, skipping of exon 51 would have the potential to
treat only 13% of DMD patients with genomic deletions on the
Leiden DMD database ( 24 ) . The continued development and
analysis of AOs for the targeting of other DMD exons are, there-
fore, vital. There have been a number of studies published describ-
ing the screens of large numbers of AOs of various chemistries for
the targeted skipping of certain exons of the DMD gene ( 25– 28 ) .
These studies collectively used a number of tools in the design of
AOs used (see Chapter 8 ). However, it should be noted that
designing an AO to have all of the desired properties will not nec-
essarily guarantee bioactivity. The empirical analysis of designed
AOs is still essential.
In this protocol, we outline the hybridization assay used to
determine target sequences open to binding by PMOs, and detail
the functional analysis (nested RT-PCR) used to assess bioactivity
of PMOs for the targeted skipping of exons on the human DMD
gene in normal human skeletal muscle cells.

Page 3

145 10 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…

1. Source of genomic DNA from the species of interest. This will
ideally be in the form of a YAC or BAC clone, but total genomic
DNA may be used.
2. Gentra Puregene Cell kit.
3. PCR primers designed to amplify genomic DNA across the
exon of interest, together with ~500 nt of upstream and down-
stream introns. The “forward” (+ strand) primer used for
amplifi cation should incorporate a minimal T7 RNA polymerase
promoter sequence (5 ¢ -TAATACGACTCACTATAGG-3 ¢ ) so
that PCR products can be used as templates for production of
pre-mRNA by in vitro transcription.
4. 2× PCR Master Mix with cresol red.
5. Access to PCR machine.
6. Agarose gel electrophoresis equipment (see Subheading 2.2 ).
7. QIAquick gel extraction kit or similar kit to isolate DNA frag-
ments from agarose gels.
8. Kit for T7 in vitro transcription, e.g. mMESSAGE mMA-
CHINE kit.
9. TURBO DNase.
10. MEGAclear kit.
11. Hexamer hybridization array screen.
12. Hybridization reaction buffer: 50 mM Tris–Cl, 5 mM MgCl 2 ,
50 mM KCl, 5 mM DTT (pH 8.5).
13. ExpandRT RNA-dependent DNA polymerase.
14. Hybridization wash buffer: 100 mM NaCl, 0.1% SDS.
15. Access to a primer design software, such as VectorNTI
(Invitrogen Ltd., Paisley, Renfrewshire, UK), or use software
freely available online (e.g. primer3: http:/ /frodo.wi.mit.edu/
primer3/input.htm ).
1. Custom PMOs (at 1 mM). Store in aliquots at −20°C.
2. Complementary DNA leash (at 200 m M). Store in aliquots
at −20°C.
3. RNase-, DNase-free sterile water.
4. Sterile 10× PBS.
1. Horizontal gel electrophoresis equipment.
2. UltraPure TM Agarose.
3. 5× TBE buffer.
2. Materials
2.1. Hybridization
Array Analysis
2.2. Annealing PMOs
and Leashes
2.3. Verifi cation
of Annealing of PMO
to Leash

Page 4

146L.J. Popplewell et al.
4. Ethidium bromide (10 mg/ml).
5. 5× DNA loading buffer.
6. DNA Hyperladder V.
1. Basic cell culturing equipment—access to 37°C, 5% CO 2 incu-
bator, Class 2 microbiological safety cabinet, 37°C water bath,
microscope, haemocytometer.
2. Source of normal human skeletal muscle primary cells (stored
in vapour phase of a liquid nitrogen storage facility).
3. Skeletal muscle cell growth medium.
4. Skeletal muscle cell growth medium supplement mix. Final
concentrations of growth factors in the complete medium are
as follows: 5% fetal calf serum (FCS), 50 m g/ml fetuin, 1 ng/ml
basic fi broblast factor, 10 ng/ml epidermal growth factor,
10 ng/ml insulin, 50 ng/ml amphotericin B, 50 m g/ml gen-
tamicin, 0.4 m g/ml dexamethasone.
5. Skeletal muscle cell differentiation medium.
6. Skeletal muscle cell differentiation medium supplement mix.
Final concentrations of growth factors in the complete medium
are as follows: 10 ng/ml insulin, 50 ng/ml amphotericin B,
50 m g/ml gentamicin.
7. Dulbecco’s modifi ed Eagle’s medium.
8. Sterile-fi ltered, cell culture-tested 200 mM L -glutamine. Store
frozen at −20°C in 5.5-ml aliquots.
9. Fetal bovine serum, certifi ed heat inactivated. Store at −20°C
in 50-ml aliquots.
10. ECM gel from Holm-Swarm murine sarcoma. Defrost over-
night at 4°C, then aliquot into a 1-ml aliquot, and store
at −20°C.
11. 0.25% Trypsin/EDTA. Store frozen at −20°C in 5-ml aliquots.
12. Mr Frosty cryo-freezing container or similar, which, when
fi lled with isopropanol, allows a cooling rate of 1°C per minute
in a −80°C freezer.
1. Skeletal muscle cell differentiation medium (e.g. from
Promocell).
2. Skeletal muscle cell differentiation medium supplement mix
(e.g. from Promocell).
3. Dulbecco’s modifi ed Eagle’s medium.
4. 5.5-ml aliquots of sterile-fi ltered,
200 mM L -glutamine.
5. Lipofectin transfection reagent.
cell culture-tested
2.4. Culturing Normal
Human Skeletal
Muscle Primary Cells
2.5. Transfection
Assays

Page 5

14710 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
1. QIAshredder kit.
2. Kit for RNA extraction, e.g. RNeasy Mini Kit.
1. GeneScript RT-PCR system kit.
2. 2× PCR Master Mix with cresol red.
3. Primers designed over exon–exon junctions to ensure that
genomic DNA is not amplifi ed.
4. RNA template harvested
Subheading 2.6 ).
from cultured cells (see
1. Horizontal gel electrophoresis equipment.
2. UltraPure TM Agarose.
3. 5× TBE buffer.
4. Ethidium bromide (10 mg/ml).
5. DNA Hyperladder IV.
6. GeneTools software available at http://www.syngene.com/
genetools/ .
1. Kit to isolate PCR fragments from agarose gels, e.g. QIAquick
gel extraction Kit.
2. Electrophoretically separated skipped and full-length products
on ethidium bromide agarose gels.
3. Access to Nanodrop to measure RNA concentration
spectrophotometrically.
4. Use of commercial company (e.g. Eurofi ns MWG Operon) to
sequence products to confi rm the correct skip.

1. Prepare the template for mRNA production. Using suitable
primer design software, design PCR primers that are situated
in the genomic DNA approximately 500 bp upstream and
downstream of the exon of interest. The “forward” primer,
located upstream of the target exon, should have a minimal
T7 RNA polymerase promoter sequence added to its 5 ¢
end. This sequence has been defi ned as 5 ¢ -TAATACGACT
CACTATAGG-3 ¢ , and means that once the PCR product has
been generated and purifi ed it can be used as a template for
in vitro transcription.
Set up a PCR reaction using 100–250 ng of genomic
DNA. This would ideally be in the form of a YAC or BAC
clone, which are now available for a wide variety of species
2.6. RNA Extraction
2.7. Nested RT-PCR
Analysis
2.8. Agarose Gel
Electrophoresis
and Densitometry
2.9. Sequencing
of PCR Products
3. Methods
3.1. Hybridization
Array Analysis

Page 6

148L.J. Popplewell et al.
from a number of sources. If this is not possible, total genomic
DNA can be extracted from cultured cells from the species of
interest using a kit, such as the Gentra Puregene Cell kit, fol-
lowing the manufacturer’s instructions closely.
2. Purify the PCR product, which is approximately 1.2 kb in
length, from an agarose gel following electrophoresis (see
Subheadings 3.10 and 3.11 ).
3. Prepare pre-mRNA using the mMESSAGE mMACHINE kit
as follows:
Thaw the frozen reagents of the kit. Add the reagents in the
following order to a microcentrifuge tube at room tempera-
ture (RT):
(a) Nuclease-free water to make fi nal reaction volume 20 m l
(b) 10 m l 2× NTP/CAP
(c) 2 m l 10× Reaction buffer
(d) 0.1–0.2 m g PCR product template
(e) 2 m l Enzyme Mix
Flick the tube or pipette the mixture up and down gently, and
then microfuge tube briefl y to collect the reaction mixture at
the bottom of the tube. Incubate at 37°C for 1 h (see Note 1).
Add 1 m l TURBO DNase, mix well, and incubate for 15 min
at 37°C. This removes the DNA template from the reaction.
Use a kit, such as Ambion MEGAclear, to purify the RNA tran-
script from unincorporated nucleotides, DNase degradation
products, enzymes, and salts. Quantify the yield of RNA by
reading absorbance at 260 nm on a Nanodrop. Store RNA at
−80°C until ready for use or continue with step 4.
4. Incubate the synthetic pre-mRNA on an immobilized array of
oligonucleotides comprising all possible sequences of six nucle-
otides in length (4,096 oligonucleotides in total) in hybridiza-
tion reaction buffer. The hybridization of the pre-mRNA to
the array is detected by the incorporation of dye-labelled
ddNTPs by the action of an RNA-dependent DNA polymerase
(reverse transcriptase), such as ExpandRT.
5. Wash off RNA, enzyme, and any excess labelled dNTPs using
hybridization wash buffer. Those oligonucleotides to which
labelled dNTPs have been added can then be detected by fl uo-
rescence. From the position on the array of the labelled oligo-
nucleotides, the sequence is known and the complementary
accessible sequences on the applied RNA can be inferred by
use of the computer software in the system (see Note 2).
PMOs are unable to enter cells in vitro due to their lack of
charge. Charge is introduced by annealing the PMOs to com-
plementary phosphorothioate-capped oligodeoxynucleotide
3.2. Leash Design

Page 7

14910 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
leashes ( 19 ) . The leashes are based on the following design,
showing a PMO designed by ourselves to induce exon 46 skip-
ping as an example.


The complementary sequence of the PMO is 17 bases long,
irrespective of whether a 25- or 30-mer PMO is being used, and
always starts at the 5 ¢ end of the PMO. The tails of the leash are
always of the sequence “gattg” (5 ¢ –3 ¢ ) at the 5 ¢ end of the PMO
and “gtgat” (5 ¢ –3 ¢ ) at the 3 ¢ end of the PMO.
As the levels of skipped transcript are generally so small relative to
the full-length transcript, it is necessary to perform nested RT-PCR
analysis on the harvested RNA to assess exon skipping induced by
the PMOs tested. This is easy to achieve when the PCR primers are
carefully designed so that they have specifi city for either the full-
length or skipped transcripts, and all the primers have similar T m s.
Using VectorNTI from Invitrogen (or similar software program),
design primers so that in each round either the forward or reverse
primer is over an exon/exon junction. This enables discrimination
between amplifi ed contaminating genomic DNA and amplifi ed
transcribed cDNA, since the PCR products derived from genomic
DNA are much larger than intronless mRNA-derived products.
The primers should be designed so that both full-length and
skipped amplicons are amplifi ed by the same primer pair, but result
in different sized products, e.g. for exon 46 skipping, use forward
fi rst-round primer designed for exon 43/exon 44 boundary and
reverse primer designed for exon 49, and then in the second round
use forward primer designed for exon 44 and reverse primer
designed for exon 48/exon 49 boundary. This leads to amplifi ca-
tion of a full-length product containing exons 44–48 and amplifi -
cation of an exon 46-skipped product containing only exons 44,
3.3. Nested RT-PCR
Primer Design

Page 8

150L.J. Popplewell et al.
45, 47, and 48. The products, being 156 bp different in size (i.e.
the size of exon 46), should be easily distinguishable on agarose
gel electrophoresis (see Fig. 1 ). General guidelines to consider
when designing primers for (RT-) PCR are to design the primers
to have a C or A at the 3 ¢ end, a GC content of ~50–60%, similar
T m s for primer pairs, and a T m greater than 55°C as calculated by
G + C rule and to avoid any internal secondary structure and poten-
tial primer-dimer formation.
PMOs are unable to enter cells in vitro due to their lack of charge.
Charge is introduced by annealing the PMOs to complementary
phosphorothioate-capped oligodeoxynucleotide leashes ( 23 ) .
Prepare annealed leash/PMO stocks at 100 m M in 50- m l aliquots
as follows.
1. Pipette 12.5 m l 10× PBS into PCR tube.
2. Add 7.5 m l RNase-, DNase-free water.
3. Add 25 m l leash (200 m M) and mix gently.
4. Add 5 m l PMO (1 mM) and mix gently.
5. Run on thermocycler program as follows: 95°C for 5 min,
85°C for 1 min, 75°C for 1 min, 65°C for 5 min, 55°C for
1 min, 45°C for 1 min, 35°C for 5 min, and 25°C for 1 min;
hold at 15°C.
6. Store at 4°C.
1. Pipette 1 m l leashed PMO (100 m M) into one tube, 1 m l of 2×
dilution of leash stock (200 m M) into another tube, and 1 m l of
10× dilution of PMO stock (1 mM) into a third tube.
2. Add 2 m l 1× PBS to each tube.
3. Add 1 m l water to each tube.
4. Incubate at 37°C for 30 min.
5. Add 1 m l of 5× DNA loading buffer.
6. Load total volume onto 3% agarose gel in TBE with 0.5 m g/ml
ethidium bromide (see Subheading 3.10 for details of agarose
gel electrophoresis).
3.4. Annealing PMOs
and Leashes
3.5. Verifi cation
of Annealing of PMO
to Leash
Fig. 1. Comparison of bioactivity of PMOs targeted to exon 46 in normal hSkMCs. Normal human skeletal myoblasts were
transfected with PMOs indicated at 500 nM using lipofectin (1:4). RNA was harvested after 24 h and subjected to nested
RT-PCR and products visualized by agarose gel electrophoresis. The positions of full-length product (exons 44–48) and
skipped product (exons 44, 45, 47, and 48) are indicated, differing in size by 156 bp (i.e. size of exon 46). The higher bio-
activity of h46A30/2 and h46A30/4, relative to the other PMOs tested, is clearly evident.

Page 9

15110 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
7. Run samples against 7.5 m l of Hyperladder V.
8. Visualize products with UV light.
An increase in size should be evident in the leashed PMO lanes
compared to leash alone if the PMO and leash have hybridized
effectively (as shown in Fig. 2 ).
1. Thaw the growth/differentiation medium supplement mix
between 15 and 25°C.
2. Remove the safety seal and open the basal growth medium in
a microbiological safety cabinet.
3. Carefully open the screwtop of the supplement and transfer the
contents with a 25-ml sterile pipette to the medium. Ensure
that the contents disperse and dissolve immediately into the
medium. Do not touch the sides of the bottle neck with the
pipette tip as this could lead to microbial contamination.
4. Note the date of addition of the supplement mix on the
bottle.
5. Close the bottle and swirl gently until a homogeneous mixture
is formed. The complete medium should be stored in the dark
at 4°C for up to 6 weeks; only the volume required should be
pre-equilibrated before use rather than the whole bottle.
1. Prior to thawing cells, supplement medium as described above.
Using a microbiological safety cabinet and standard cell cul-
ture techniques, pre-equilibrate a 75-cm 2 culture fl ask with
14 ml of growth medium at 37°C in a 5% CO 2 incubator.
2. Transfer the cryovial containing ~5 × 10 5 normal human skel-
etal muscle cells in a 1-ml volume quickly from liquid nitrogen
to a 37-C water bath, ensuring that the cap is not fully sub-
merged. NB: Wear appropriate safety equipment, i.e. goggles,
3.6. Culturing
of Normal Human
Skeletal Muscle
Primary Cells
3.6.1. Preparation
of Growth and
Differentiation Media
3.6.2. Initiation
of Proliferating Cultures
from Cryopreserved Cells
Fig. 2. Verifi cation of annealing of leash to PMO. The larger size of the leashed PMOs (as
indicated by thick arrow ) relative to the size of the leash alone (as indicated by thin arrow )
indicates successful annealing of complementary leash to PMO. It is interesting to note
that PMO alone, being uncharged, does not electrophorese into the agarose gel.

Page 10

152L.J. Popplewell et al.
gloves, and lab coat. Swirl the vial gently but rapidly for
1–2 min until only a small piece of ice (grain of rice size) is left.
Wipe the vial dry and transfer the vial on ice to the microbio-
logical safety cabinet.
3. Rinse the vial with alcohol, and wipe to remove the excess.
Open the vial and gently pipette the cell suspension up and
down to evenly suspend the cells.
4. Pipette the cells into the prepared culture fl ask in an arc on the
surface of the medium, and gently swirl the medium to dis-
perse the cells for even growth.
5. Examine the cells microscopically to check even distribution of
the cells in the fl ask, and transfer to the 5% CO 2 incubator. Do
not disturb the culture for the next 16 h to allow cell
attachment.
6. After a maximum of 24 h, examine the culture microscopically
to check whether seeding has been successful. To remove the
DMSO present in the freezing mix, replace the medium with
15 ml of pre-equilibrated fresh medium. Be careful to run the
medium over a cell-free surface of the fl ask and never over the
cell layer as this may dislodge the cells.
7. Return the cells to the incubator, re-feed with fresh media
every 48 h, and subculture when the cells reach 60–80% con-
fl uence and while they are still actively dividing.
Once the cells have reached 80% confl uence, passage in a biological
safety cabinet as described below to provide cells for transfection in
6-well plates, proliferating culture in a 75-cm 2 fl ask, and one vial of
cells for cryopreservation.
1. Prepare one 75-cm 2 fl ask by equilibrating 14 ml fresh supple-
mented growth medium for at least 30 min in the 5% CO 2
incubator.
2. Pre-warm a 50 ml of DMEM supplemented with L -glutamine,
50 ml supplemented growth medium, and 20 ml supplemented
growth medium with 10% FCS which acts as trypsin blocking
solution (i.e. add 2.5 ml FCS to 47.5 ml supplemented growth
medium) in a 37°C water bath.
3. Aliquot out 18 ml ice-cold DMEM supplemented with
L -glutamine onto ice. Quickly defrost 2 × 1-ml aliquots of ECM
gel in a 37-C water bath for 1–2 min until only a small piece of
ice (grain of rice size) is left. Add the ECM gel to ice-cold DMEM,
pipette to mix, and then aliquot out onto 4 × 6-well plates.
Incubate in 5% CO 2 incubator for at least 45 min before use.
4. To passage the cells, aspirate the old media and rinse the cell
layer gently with 15 ml pre-warmed DMEM supplemented
with L -glutamine to remove traces of FCS.
3.6.3. Subculture
of Proliferating Cultures

Page 11

153 10 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
5. Add 1 ml trypsin/EDTA (defrosted to RT), ensuring that the
entire surface of the cell sheet is covered.
6. Monitor trypsinization microscopically, tapping the fl ask
sharply to detach the cells once they have rounded up. This
should take around a minute (see Note 3).
7. Quickly add 15 ml pre-warmed trypsin blocking solution (i.e.
supplemented growth medium with 10% FCS) and transfer the
cells to a centrifuge tube. Rinse the fl ask with 5 ml DMEM
supplemented with L -glutamine to collect residual cells and
add to the centrifuge tube. Centrifuge for 5 min at 220 × g .
8. Discard the supernatant and gently re-suspend the cell pellet
in 2 ml pre-warmed supplemented growth medium with
10% FCS.
9. Perform a cell count with a haemocytometer and adjust cell
volume to give 500,000 cells/ml. Typically, this gives a fi nal
volume of cell suspension of 6 ml.
10. Remove 0.9 ml of the cell suspension into a cryovial, add
100 m l DMSO (to give freezing mix with 10% FCS and 10%
DMSO), and pipette to mix. Freeze cryovials overnight in a
suitable cryo-freezing container (e.g. “Mr Frosty” container)
and then transfer to the vapour phase of a liquid nitrogen stor-
age facility.
11. Remove 1 ml of the cell suspension into the 75-cm 2 fl ask con-
taining 14 ml of pre-equilibrated supplemented growth
medium, and gently swirl to mix cells into the media. Check
fl ask microscopically for even seeding of cells and transfer to
5% CO 2 incubator. Feed culture every 48 h with fresh growth
media, and repeat subculture procedure when they reach
60–80% confl uence (see Note 4).
12. Remove the 4× 6-well plates from the 5% incubator and care-
fully aspirate the ECM gel. Aliquot 1 ml of pre-warmed sup-
plemented growth media into each well. Dilute the remaining
cell suspension to a total volume of 24 ml with pre-warmed
growth media. Mix thoroughly by pipetting and aliquot care-
fully 1 ml of cell suspension into each well. Gently rock the
plates to ensure mixing of cell with the media. Check plates
microscopically for even seeding of the cells and transfer to 5%
CO 2 incubator. The cells are ready for transfection when they
reach 80% confl uence (typically, 48 h after plating out). Feed
every 48 h if required. See Notes 5–7.
Once the cells in the 6-well plates have reached 80% confl uence,
they are ready for transfection. If the cells are allowed to pass 80%
confl uence, transfection success is compromised. The method
described here is for transfections with lipofectin as transfection
reagent. Lipofectin is used in the absence of antibiotics to avoid
3.7. Transfection
Assays

Page 12

154L.J. Popplewell et al.
cell death, and performs optimally in media without serum. As a
negative control, leashed PMOs are used without lipofectin and to
assess toxicity of the transfection reagent, diluted lipofectin should
be used alone, and blank controls of DMEM alone included for
reference. The protocol below is for the transfection of 24 wells
(i.e. four 6-well plates). Adjust volumes given accordingly for big-
ger or smaller experiments.
1. Pre-warm 50 ml of supplemented differentiation media and
100 ml DMEM supplemented with L -glutamine.
2. Aspirate growth media from cells, replace with 2 ml of differ-
entiation media, and transfer to a 5% incubator for 1 h.
3. Dilute the leashed PMO in DMEM supplemented with
L -glutamine to give the required fi nal concentration in the
transfection mix, e.g. for a fi nal concentration of 500 nM in a
1-ml transfection volume, use 15.5 m l of 100 m M of stock
leashed PMO and 294.5 m l of DMEM to give enough for 3
wells.
4. Mix the lipofectin before use and then dilute to give a fi nal
ratio of 1:4 ( m g DNA: m l lipofectin). That is, for 1 ml of 500
nM of leashed PMO fi nal concentration and to have suffi cient
for 24 wells, use 252 m l lipofectin with 2.016 ml DMEM. Let
stand for 30 min at RT. Adjust the dilution of lipofection to
maintain the ratio at 1:4, in relation to the fi nal concentration
of leashed PMOs used.
5. Combine the diluted lipofectin with the diluted leashed PMO,
i.e. to 310 m l of diluted leashed PMO, add 310 m l of diluted
lipofectin. Mix gently by pipetting and incubate for 10–15 min
at RT to allow complex formation.
6. Remove the differentiation medium from the cells and wash
once with 2 ml of pre-warmed DMEM supplemented with
L -glutamine. Remove the wash medium.
7. Add 0.8 ml pre-warmed DMEM supplemented with L -glutamine
to each well. Add 200 m l of leashed PMO/lipofectin mix to
respective wells, typically setting up three reps of each, and mix
gently by rocking the plates.
8. Incubate the cells at 37°C in a 5% CO 2 incubator for 4 h.
9. Replace the transfection mix with 2 ml of pre-warmed supple-
mented differentiation media.
10. Incubate the cells at 37°C in a 5% CO 2 incubator and harvest
RNA at set time points following transfection. This would
typically be 24 h after transfection, but can be extended to
anything up to 14 days, with feeding of the cells every 48 h
with 2 ml of fresh pre-warmed supplemented differentiation
media. See Notes 8–11.

Page 13

155 10 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
RNA is extracted from the cells using the RNeasy mini kit and
QIAshredder columns at predetermined time points after transfec-
tion of the cells with leashed PMOs. Typically, this is 24 h after
transfection, but for studies examining the persistence of exon
skipping, RNA should be harvested at days 1, 2, 3, 7, 10, and 14.
The cultures should be fed every 2 days to ensure their survival. It
should be noted that to prevent damage and possible contamina-
tion of harvested RNA all steps should be carried out carefully, but
with speed. Gloves should be worn at all times and samples should
be kept on ice as much as possible.
1. Carefully remove cell culture medium supernatant from cells
by aspiration, and ensure that all supernatant is removed. The
medium inhibits cell lysis and dilutes the lysate and thereby
affects binding of the RNA to RNeasy silica-gel membrane, so
affecting RNA yield.
2. Add 350 m l of buffer RLT to each well of the 6-well plate that
is to be harvested. Carefully pipette the buffer a number of times
over the surface of the well to ensure that all cells are lysed.
3. To ensure complete homogenization of the cells so that the
highest yield possible of RNA is achieved and clogging of the
RNeasy mini column is prevented, pipette the cell lysate directly
onto a QIAshredder spin column placed in a 2-ml collection
tube and centrifuge for 2 min at maximum speed. Remove
QIAshredder column and discard.
4. Homogenized cell lysates in buffer RLT can be stored at −70°C
for several months. Simply cap, label, and freeze. To process
frozen lysates, thaw samples at 37°C for 15–20 min to dissolve
salts. If any insoluble material is still visible, centrifuge for
5 min at 3,000–5,000 × g . Transfer supernatant to a new sterile
microcentrifuge tube, and continue with step 5.
5. Add 350 m l of 70% ethanol to homogenized lysate, and mix
well by pipetting.
6. Apply up to 700 m l of the sample, including any precipitate
that may have formed to an RNeasy mini column placed in a
2-ml collection tube. Close the tube gently and spin for 15 s at
³ 8,000 × g ( ³ 10,000 rpm in a microfuge). Discard the fl ow
through.
7. Add 700 m l Buffer RW1 to the RNeasy column. Close the tube
gently, and centrifuge for 15 s at ³ 8,000 × g . Discard the fl ow
through.
8. Pipette 500 m l Buffer RPE onto the RNeasy column. Buffer
RPE is supplied as a concentrate. Before using for the fi rst
time, add four volumes of ethanol (96–100%) (i.e. 44 ml of
ethanol to 11 ml of Buffer RPE concentrate) to obtain a work-
ing solution.
3.8. RNA Extraction

Page 14

156L.J. Popplewell et al.
9. Close the tube gently and centrifuge for 15 s at ³ 8,000 × g to
wash the column. Discard the fl ow through.
10. Add another 500 m l Buffer RPE to the RNeasy column. Close
the tube gently, and centrifuge for 2 min at ³ 8,000 × g to dry
the RNeasy silica-gel membrane.
11. To elute the RNA from the column, carefully transfer the
RNeasy column, ensuring no contact of the column with the
fl ow through, to a new 1.5-ml collection tube. Pipette 30 m l
RNase-free water directly onto the RNeasy silica-gel mem-
brane. Close the tube gently, and centrifuge for 1 min at
³ 8,000 × g to elute.
12. Assess the concentration of RNA spectrophotometrically at
260 nm on a Nanodrop and adjust the concentration of the
RNA to 0.5 m g/ m l with nuclease-free sterile water. Store RNA
samples at −70°C. See Notes 12 and 13.
As the levels of skipped transcript are generally so small relative to
the full-length transcript and dystrophin expression in cultured
cells is low anyway, it is necessary to perform nested RT-PCR anal-
ysis on the harvested RNA. This is easy to achieve when the PCR
primers are carefully designed so that they have specifi city for either
the full-length or skipped transcripts and all the primers have simi-
lar T m s (see Subheading 3.3 ). Using a sample that should only con-
tain full-length transcript (e.g. lipofectin alone) and a sample that,
on the basis of previously published work and strength of PMO
design tools used, should contain skipped transcript along with
full-length transcript, ascertain the optimal temperature for the
PCR by performing a temperature-gradient PCR for both the fi rst
round and the second round. This is the temperature that gives a
clean full-length product in the lipofectin-alone RNA sample and
no skipped product, and a full-length and skipped product, in the
PMO-transfected RNA sample. However, for reassurance, you may
wish to order from GeneArt RNA sequences that correspond to
the full-length and, perhaps most importantly, skipped transcripts.
If there is no facility for performing temperature gradient, perform
the nested RT-PCR on a full-length and skipped sample at an
annealing temperature just below the Tm for the primers used.
Adjust the annealing temperature subsequently if required. The
methodology described below is the one used by ourselves for ana-
lyzing RNA samples harvested from transfected cells on four 6-well
plates (i.e. 24 samples) for the targeted skipping of exon 46.
1. Thaw all components of the GeneScript RT PCR kit and keep
on ice. Wear gloves throughout to avoid any contamination
with RNases.
2. Briefl y vertex and microfuge all reagents before preparing the
reactions.
3.9. Nested RT-PCR
Analysis
3.9.1. First-Round RT-PCR

Page 15

15710 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate…
3. Set up two master mixes, prepared in two separate RNase- and
DNase-free microcentrifuge tubes kept on ice. Set up as out-
lined in Tables 1 and 2 .
4. Carefully pipette 2 m l of each RNA to be tested (containing
1 m g of RNA) into the bottom of a 0.2-ml thin-wall PCR tube
on ice using a fresh tube for each sample. It is important to
include a tube with 2 m l of RNAse-free sterile water to rule out
the possibility of cross-contamination of samples (25 tubes
in total).
5. Vortex master mix 1 (Table 1 ) and carefully pipette 10 m l into
each PCR tube on ice using a fresh tip for each tube to ensure
no cross-contamination of samples.
6. Vortex master mix 2 (Table 2 ) and carefully pipette 13 m l into
each PCR tube on ice using a fresh tip for each tube to ensure
no cross-contamination of samples.
Place the tubes in a thermal cycler at 45°C and cycle as follows:
for fi rst-strand cDNA synthesis, incubate at 45°C for 30 min;
for RT inactivation (otherwise, the RT enzyme can hamper
Table 1
Preparation of master mix 1
Master mix 1
Volume for 1 tube
(25 m l fi nal)
Volume for
24 tube stock
Final
concentration
Nuclease-free sterile water Up to 10 m l Up to 260 m l
5 mM dNTP mix 1 m l 26 m l 200 m M
100 m M forward outer primer 0.25 m l 6.5 m l 1 m M
100 m M reverse outer primer 0.25 m l 6.5 m l 1 m M
Table 2
Preparation of master mix 2
Master mix 2
Volume for 1 tube
(25 m l fi nal)
Volume for 24
tube stock
Final
concentration
Nuclease-free sterile water Up to 13 m l Up to 338 m l
5× RT-PCR buffer (includes 7.5 mM
MgSO 4 )
GeneScript enzyme mix (blended mix
of Accurase DNA polymerase and
MMuLV reverse transcriptase)
5 m l 130 m l 1.5 mM MgSO 4
0.25 m l 6.5 m l 0.625 U