Transcript CRISPR-Cas-9 for new therapies

CRISPR/Cas9
Breakthrough in genome editing.
The most important scientific result
of the year 2015.
“Science“,18 December 2015
I will report first about
Duchenne muscular dystrophy,
because there were serious problems
during the development of this therapy.
Then I will report how these problems
can probably be solved
in the not too distant future
by the new technique CRISPR/Cas9.
This is Christian
when he was 5 years old.
He had
Duchenne muscular dystrophy.
I found him with our
„firefly CK screening test“,
when he was 3 weeks old.
Duchenne is a hereditary disease
which affects only boys.
The disease becomes visible
when the children are 4 – 6 years old.
At the age of about 12, the boys need a wheelchair,
but otherwise they can practically lead a normal life
and also defend their interests,
as shown here at a demonstration
in Washington for more research.
The sick children are being born
without showing symptoms of their disease.
But because the largest of their 20,488 genes
has a mistake, so that an important protein
in their muscles cannot be made,
and because this hereditary disease is not yet curable,
the boys are being born with a death sentence,
that is being executed at about 25 years,
when they die by heart and respiratory failure.
About 1 in 5,000 boys has Duchenne.
Today, there are about
300,000 – 400,000 Duchenne boys
and young men living throughout the world.
The disease is caused by mutations (mistakes)
of the Dystrophin gene.
Genes are long sections of the double helix of
the genetic material.
The steps of the helix ladder contain the
genetic information, they are the genetic
letters.
The letters are chemical substances, whose
names are abbreviated as A, G, C and T
(or U).
The gene contains the information for the
production of the protein dystrophin, which
stabilizes the muscles.
The dystrophin gene is the largest of our
20.488 genes, it has 2.220.223 genetic letters.
James Watson and Francis
Crick discovered the double
helix, built a model of wire,
and described it on 25 April
1953 in the journal Nature.
The chemical name of the
double helix is desoxyribonucleic acid, abbreviated as DNA.
One of the most important characteristics
of the double helix is the
“Watson-Crick” bond of both strands.
Here, a short section of the helix is
spread out like a ladder.
The strands are hold together by the rungs
which always consist of
two different base pairs:
A-T or T-A
and
G-C or C-G
The names of the
4 genetic letters are:
A = Adenine
T = Thymine
G= Guanine
C= Cytosine
They are chemical substances, bases, which fit
exactly between the two strands.
The sequence of the letters
of the base pairs,
contains the genetic information
for the function of a living cell.
It can also be inherited.
The strands can separate
so that the information
can be read or changed.
If a base sequence must
be attached to a certain
place of the DNA to
obtain an effect, this
piece of the DNA can be
added. It then moves to
the desired place and
connects there so that the
correct base pairs are
formed by the WatsonCrick configuration.
Here, the two sequences have arrived at
the desired places of the DNA.
The sequences are complementary:
an A is always bound to a T,
and a G to a C, (or vice versa).
This works like a zipper
with 4 different teeth.
This searching and finding
is very specific and plays
a decisive role for the CRISPR method.
Dystrophin, whose mutation causes Duchenne, is part of a
complex of many proteins in the membranes of the muscle cells.
It stabilizes the muscle membranes during the contractions
of the muscles and works like a shock absorber.
To get an impression of the real sizes
of the things we are talking about:
The dystrophin gene with its 2.2 million genetic letters
is 0.75 mm long if extended.
Curled together, it fits into
a cell nucleus of 0.01 mm diameter.
A molecule of dystrophin is 0,000125 mm long.
8.000 dystrophins arranged one after the other
fit into 1 millimeter.
One gram muscle tissue
contains 114 billion dystrophin molecules.
For about 20 years work is being done on the most
promising of the other 40 other possible therapies:
Exon Skipping,
which could slow down
the degradation of the muscles.
Here is a short summary:
Of the 2.2 million genetic letters of the dystrophin gene,
only 11,000 are used
for the biosynthesis of the protein dystrophin.
They are grouped into 79 active sequences called exons.
Part of the base sequence of the dystrophin gene.
Two exons
Between the exons are introns which do not “make”
proteins.They are often much longer than the exons.
Aataaattaactttactgctcatctcattggtctgccaaattggaaagacattttaggcctcat tctcatgtt
ctaattagtctctggaggacattcatggacaattcactgttcattaatctatcgtcttccttta actttgatt
tgttcattatccttttagagtctcaaatatagaaaccaaaaattgatgtgtagtttaatgtgct tacagATGT
TGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCACATCACTCTTCCAAGTTTTG CCTCAACAA
GTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAG AACATTTTC
AGTTACATCATCAAATGCACTATTCTCAACAGgtaagtgtgtaaaggacagctactattcaagatgttttctg
ttttatatgcatttttaggtattacgtgcacatatatatataacttatatgtatatacacgtgt atatataca
aagcctaatgtatgtatatgtacaaaaagagacagactaaaccttttcaattttgcttcattga ctacaatat
tcatttcaaaacatattttaaaaatactattcatatgtaccatatcataggaatatagaacata atatattaa
atttccattgtgtaacacacaatgaaatgaaaggcaaacttacgtacatattaaactctaaaat cttcatcaa
ctagaacaaagagctagagaaagattaaaggcaaatacagtttttaatcattaaaaaatacttt atgacaata
tgatgtacaaagtcagattttgattattcaggataacaattttgaaaatagaaaagtgttttaa actatttca
aataaacaataaagaaaaacattgatgaaattcttcgttacataattgtatagaatttagtggggttcttcca
tgacattggcttgttctttctctcaacagtgggtggtttggatgttttcctatgctttctcagg cacaaacaa
cagtgaagaaacctttagcaacatttctgctgaatgtgtggagagctcttaggccaaactctct cactttgct
gagctagctctgtattagcttatgaaatttttagtcctttccagggtatggacctgatgaattc tgtttttat
ttactttggatttagagaaaattgcttatggatgcctgtttttgccggattgaagagtaccatc ccatataaa
tgttggagttgtgttactttggaatcctcatggctaaatagcatgctcatatcttcccagatca tttactctc
caacatgatattaagtgccttcattctgggagtataccaatttcgaatatcatttatatatgtt tagtattaa
acattgaatctcaccatttccatttaatgttctgttttctaccatgttggaaagtagtcctttc gggttactt
atggtttttccccctcctctctatccactcccccaaacccttctctgcagATCACGGTCAGTCTAGCACAGGG
ATATGAGAGAACTTCTTCCCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTAT GTCACCACC
TCTGACCCTACACGGAGCCCATTTCCTTCACAGgtctgtcaacatttactctctgttgtacaaaccagagaac
tgcttccaagataatctaacactgcttttacttgcttgaatttttcagtgccttttatctcctc gtgaagagc
tggtttgtttttccaagttttattgattccctcatgtgattctgctttgttattgagtcttctc taggtttac
The borders of the 79 exons of the normal
dystrophin gene fit exactly into each other.
15
16
28
29
17
41
42
54
55
68
3 4
2
1
69
30
5
6
18 19
20
31
43
56
70
33
32
44
57
7172 73
45
58
74
8
7
21
22
34
35
46
59
75
10
9
47
23
36
48
24 25
37
49 50
60 61 6263 64
76 77 78
12
11
13 14
40
38 39
51
65
27
26
52
66
79
100 nt
When the 4 exons
47, 48, 49 and 50 are
missing after a mutation…..
53
67
Stop sign in exon 51
1
15
16
28
29
17
41
42
54
55
68
3 4
2
69
100 nt
30
5
6
18 19
20
31
43
56
70
33
32
44
57
7172 73
45
58
74
8
7
10
9
21
22
34
35
23
36
24 25
37
75
60 61 6263 64
76 77 78
13 14
65
27
26
40
38 39
51
46
59
12
11
52
66
53
67
79
…then the flanking exons 46 and 51 do not fit together
and the child has Duchenne dystrophy, because a stop
sign appears in exon 51.
To make them fit again, exon 51 has
to be skipped, so that its information is not used
any more for the biosynthesis of the dystrophin.
1
15
16
28
29
17
41
42
54
55
68
3 4
2
69
30
5
6
18 19
20
31
43
56
70
33
32
44
57
7172 73
45
58
74
8
7
10
9
21
22
34
35
23
36
24 25
37
46
59
75
12
11
76 77 78
65
27
26
40
38 39
51
60 61 6263 64
13 14
52
66
53
67
79
100 nt
… then fit exactly into
The left end of exon 52 will
the right end of exon 46.
1
15
16
28
29
17
41
42
54
55
68
3 4
2
69
100 nt
30
5
6
18 19
20
31
43
56
70
33
32
44
57
7172 73
45
58
74
8
7
10
9
21
22
34
35
23
36
12
11
24 25
37
75
27
26
40
38 39
52
46
59
13 14
60 61 6263 64
76 77 78
65
66
53
67
79
Then, no stop sign appears any more, the dystrophin is
produced again, but it is shorter than normal.
This leads to the the more benign form of the disease,
Becker dystrophy.
Therefore, exon skipping is not a cure, but a therapy, that
would only slow down the progress of the disease.
The drug which can skip exon 51, is an antisense-oligoribonucleotide with a sequence of 20 genetic letters. It connects to
the complementary sequence in exon 51 using the WatsonCrick configuratiion and blocks the entire exon. This is then
skipped and not used any more for the synthesis of dystrophin.
Here are the two sequences (instead of T, the similar base
uridine, U, is used.)
UCUUUACGGUAGAAGGAACU
AGAAAUGCCAUCUUCCUUGA
The blue sequence is the Duchenne drug Kyndrisa. It consists of 700 atoms. The red sequence is the complementary
sequence in exon 51.
A is always opposite U, and G opposite C.
13 % of all Duchenne patients need this skipping of exon 51,
the largest group of 80% of all Duchenne patients,
which need all other kinds of exon skipping.
To prove the effectiveness of exon 51 skipping,
a series of clinical studies were performed since 2004.
Finally in 2013/14,
the large pharmaceutical company Glaxo-Smith-Kline
performed the decisive phase-III trial
with 186 patients in 42 countries,
double-blind, for one year.
Unfortunately, the result compared with the placebo group
was not significant, not sufficiently better.
Therefore, the US Federal Drug Agency FDA
informed the now responsible
company BioMarin in San Rafael in California
on 14 January 2016, that:
“The application is not ready for approval
in its present form.”
This means that for many Duchenne boys,
an exon skipping therapy will come too late.
I will now start the second part of my presentation.
The scientific result declared by the journal Science
Breakthrough of the Year 2015
about the already mentioned
CRISPR/Cas9 technique,
which perhaps could lead to
a Duchenne therapy in the near future.
The most important scientists to mention here
are two ladies and one man.
Emmanuelle Charpentier of
the Max-Planck-Institute for
Infection Biology in Berlin and
Jennifer Doudna of the
University of California in
Berkeley. Each of them got a
3-Million- DollarBreakthrough-Price from a
group of billionaires in
November 2015.
And on 14 March 2016 they
will get the Paul-Ehrlich-Award
2016 in Frankfurt’s
Paulskirche (100,000 Euro).
Feng Zhang of Harvard’s Broad
Institute and the MIT in
Cambridge, Mass.
The two ladies worked with
purified substances while Dr.
Zhang worked with entire cells.
Therefore Dr. Zhang
got the patent for CRISP/Cas9.
And now there are big
discussions about who was first
between their employers.
CRISPR is the abbreviation for:
Clustered regularly interspaced short palindromic repeats,
and Cas9 means CRISPR associated nuclease.
(Nobody knows this by heart!)
The CRISPR-DNA-sequence with the
Cas9-enzyme was found in bacteria,
which use them to defend themselves against viruses.
They do this by cutting both of the strands
of the viral DNA
and thus cut the viruses into pieces.
In 2012, Jennifer – later with Emmanuelle – and Feng
got the idea to modify this ingenious method so that
also other double helix DNAs could be cut completely
at exactly defined sites.
You only have to add a nucleic acid sequence
to the CRISPR protein that fits exactly
to the complimentary gene sequence to be cut.
CRISPR then searches for this sequence to be cut
among the 3 billion genetic letters of all chromosomes,
attaches to it, and the Cas9 cuts the DNA there.
The next slide shows in a simplified way
how this is done.
The protein of the The guide RNA, which searches for the site
CRISPR system in the gene to be cut,
attaches itself there by WC binding,
and cuts both strands of the DNA.
The DNA
.
of the gene
The aim of the new CRISPR technique
was immediately to repair DNA with mutations
– with mistakes – that cause hereditary diseases.
Duchenne muscular dystrophy was
one of the first diseases, for which this was tried
and described on 22 January 2016
in 3 publications in the journal Science.
I am explaining the work of Charles Gersbach’s team
at Duke University in Durham, North Carolina.
At first this new therapy was not tried on Duchenne boys,
but on the mdx mouse, which also has Duchenne.
In this small animal, exon 23 has to be removed,
so that its dystrophin can be made again.
The CRISPR/Cas9 system was transported
into the muscles of the mouse
with harmless adeno viruses.
The CRISPR then cuts the DNA to the right and to the left
of exon 23 and the reparation system of the muscle cells
joins the two ends of the DNA again.
On these pictures
You can see that exon 23
was really cut out:
The two used CRISPRs with their
guide sequences,
the exon chain with
the cut out exon 23,
and the proof
that in the mouse
exon 24 follows
directly after exon 22.
I am showing one of the mdx mice
which have been treated with the CRISPR system.
Here comes Gustav, quite slowly,
showing how he walked before the treatment.
And after the treatment he can run quite fast
and his heart problems have gone also.
Thus it was proved that the new technique
really can cure a hereditary disease.
But for the time being, only a small animal.
A child is much larger.
Transporting the CRISPR systems with
its Cas9 proteins into the muscles of a child
requires a working gene therapy technique.
The development for humans, however,
is only at the beginning.
Therefore, it will still take several years,
until a therapy will be available
for Duchenne muscular dystrophy
using the CRISPR/Cas9 system.
Thus the development of exon skipping has to continue and
also all the other possible Duchenne therapies.
At the end: Something important:
Exon skipping changes only the information of a gene.
The gene itself will not be changed.
Therefore, the medical effect will not be inherited.
With the CRISPR technique
the gene itself can be changed.
.
Therefore the effect can also be inherited.
Discussions about the new technique
are already going on intensely.
Its use should be strictly controlled.
Beneficial consequences are for instance:
Many of the rare hereditary diseases
– if their genetic causes are known in detail –
will become treatable with CRISPR.
With “gene drive”, whole populations of harmful insects
can be eradicated within a short time.
For instance: the anopheles mosquitoes and with them
malaria could be eliminated within a few years.
That was 20 years of research
described in about 45 minutes!
Thank your
for your
attention.