Why study RNA?

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Why study RNA?
Gene expression regulation
post-transcriptional regulation polyadenylation
splicing editing transport translation stability
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Therapeutic RNAs New RNA functions and
activities Antisense Aptamers Ribozymes Modifyin
g RNAs RNA interference
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antisense RNAs
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Correction of splicing of ?-globin pre-mRNA
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Aptamers
RNA molecules capable of binding specifically to
target proteins
- epitope recognition similar to monoclonal
antibodies - they can be developed more rapidly
and at lower cost - use as protein or enzyme
inhibitors in vivo
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Ribozymes
RNA molecules capable of recognizing other RNA
molecules and to cleave them
- sequence specific (point mutations can be
distinguished) - sensitive - in vivo, they drive
the target RNA to the degradative pathway
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RNA interference
siRNAs - short (21-23 bp)double stranded RNA
mole-cules able to direct the target RNA to a
discard pathway
- sequence specific - very effective - the
target RNA is degraded
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Inhibition of splicing by modifying snoRNAs
CH3
E3
E2
E1
methylating snoRNA
The 2-O-methylation of the branch site blocks
the first step of splicing The 2-O-methylation
of the 3 splice site blocks the 2nd step of
splicing
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- RNA molecules can interfere with gene
expression in a sequence-specific way
Advantages of RNA-base gene therapy
- The specificity is extremely high and can
be obtained with molecules of low complexity
- Non-immunogenic
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Therapeutic RNAs should be stably expressed in
order to obtain a long term activity
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Requirements for an effective therapeutic RNA
- Efficient expression polIII, polII promoters,
LTR
- Stability insert into stable RNAs
- Structure pairing regions exposed
- Subcellular compartmentalization co-localizatio
n with the target
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Genes for small RNAs as vectors for the delivery
of therapeutic RNAs
RNA localization function
U1 nucleoplasm splicing
U2 nucleoplasm splicing
U7 nucleoplasm 3 processing of histone
pre-mRNA
U16 nucleolus site-specific methylation
of pre-rRNA
VA1 cytoplasm translation interference
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small non-coding RNAs
SIZE
of
of
SPECIES
COPIES
(nucl.)
lt10
lt300
high
small nuclear RNAs
Nucleus
(snRNAs)
small nucleolar RNAs
gt100
low
lt300
(snoRNAs)
Nucleoli
Cytoplasm
small cytoplasmic RNAs
?
?
lt100
(scRNAs)
micro RNAs
gt100
?
21-23
(mRNAs)
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Use of RNA for gene therapy of HIV- ribozymes
-- aptamers -
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- Rev is a viral protein that exports unspliced
and singly spliced HIV RNA - Rev binds to the
RRE RNA in the envelope gene through its RNA
binding motif - Rev binds to CRM1 and the
nuclear export machinery. It shuttles between the
nucleus and the cytoplasm.
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HIV alternative splicing
2-kb spliced
An
Alternative splicing
4-kb spliced
An
Alternative splicing
An
9-kb unspliced
Transcription/ Cleavage-polyadenylation
LTR
LTR
int
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Northern of HIV RNA
rev- HIV-infected
Uninfected cells
HIV-infected
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The HIV regulatory proteins Tat and Rev have a
nucleolar localization property in human cells.
However, no functional role has been attributed
to this localization. Recently it has been
demonstrated that expression of Rev induces
nucleolar relocalization of some protein factors
involved in Rev export. Because the function of
Rev is to bind HIV RNA and facilitate transport
of singly spliced and unspliced RNA to the
cytoplasm, it is likely that the nucleolus plays
a critical role in HIV-1 RNA export. As a test
for trafficking of HIV-1 RNAs into the nucleolus,
a decoy for Rev and a hammerhead ribozyme that
specifically cleaves HIV-1 RNA were inserted into
the body of the U16 small nucleolar RNA,
resulting in accumulation of the chimeric RNAs
within the nucleoli of human cells. HeLa CD4 and
T cells expressing this nucleolar localized decoy
and ribozyme exhibit dramatically suppressed
HIV-1 replication. The results presented here
suggest a trafficking of Rev and HIV-1 RNA
through the nucleoli of human cells, thus posing
a different paradigm for lentiviral RNA
processing.
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Does the HIV Rev protein has a nucleolar phase?
Rev
No
Cyt
Nu
Splicing
Transport
Env
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U16-RBE decoy competes with RRE for Rev binding
Rev
No
Cyt
Nu
HIV pre-mRNA
RRE
Rev decoy
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A
A
U
C
G
U
A
RBE
G
C
C
G
G
C
A
U
C
G
A
G
C
C
G
G
G
G
U
A
G
C
A
U
U
G
box C
U
box C
A
C
U
G
U
box D
box D
G
A
A
U
5'
AAA
3'
5'
AAA
3'
U16
U16-RBE
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.
U16-RBE expression
293
X. laevis
M
RBE
p
m
m'
1
U6 prom
A
A
A
T
T
T
T
T
T
m'
m
p
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HIV-IIIB infection of CD4 Hela cells
transfected with U16-RBE
3000
A3 RBE
?
pBabe
?
?
2000
p24 pg/ml
1000
?
?
?
?
?
0
20
40
60
80
h Post Infection
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Does HIV pre-mRNA has a nucleolar phase?
Rev
No
RRE
Cyt
Nu
RRE
RRE
Env
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A nucleolar ribozyme should prevent the
accumulation of HIV transcripts
Rev
No
IIIIII
RRE
Cyt
RRE
Nu
RRE
RRE
U16-Rz
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Anti HIV pre-mRNA ribozyme
U
G
box C
U
box C
C
A
U
G
U
box D
box D
G
A
A
U
5'
AAA
5'
3'
AAA
3'
U16
U16-Rz
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A) pBaBe puro retroviral vector
gag
SV40
puro
B) pBabe puro/U16hRz or U16hRBE Orientation 1
gag
SV40
puro
U16-Rz/RBE
U6p
C) pBabe puro/U16hRz or U16hRBE Orientation 2
gag
SV40
puro
U16-Rz/RBE
U6p
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HIV-IIIB infection of CD4 Hela cells
transfected with U16-Rz and U16-RBE
3000
A1 WT Rz
l
A2 MUT Rz
u
A3 RBE
s
u
n
pBabe
n
2000
p24 pg/ml
s
s
1000
u
n
l
l
l
s
n
u
0
20
40
60
80
h Post Infection
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Mol Ther. 2003, 8196-206. Inhibition of HIV-1
infection by lentiviral vectors expressing pol
III-promoted anti-HIV RNAs Ming-Jie Li1,
Gerhard Bauer2, Alessandro Michienzi1, Jiing-Kuan
Yee2, Nan-Sook Lee1, James Kim1, Shirley Li2,
Daniela Castanotto1, John Zaia2 and John J.
Rossi, , 1 Abstract A primary advantage of
lentiviral vectors is their ability to pass
through the nuclear envelope into the cell
nucleus thereby allowing transduction of
nondividing cells. Using HIV-based lentiviral
vectors, we delivered an anti-CCR5 ribozyme
(CCR5RZ), a nucleolar localizing TAR RNA decoy,
or Pol III-expressed siRNA genes into cultured
and primary cells. The CCR5RZ is driven by the
adenoviral VA1 Pol III promoter, while the human
U6 snRNA Pol III-transcribed TAR decoy is
embedded in a U16 snoRNA (designated U16TAR), and
the siRNAs were expressed from the human U6 Pol
III promoter. The transduction efficiencies of
these vectors ranged from 9698 in 293 cells to
1520 in primary PBMCs. A combination of the
CCR5RZ and U16TAR decoy in a single vector
backbone gave enhanced protection against HIV-1
challenge in a selective survival assay in both
primary T cells and CD34-derived monocytes. The
lentiviral vector backbone-expressed siRNAs also
showed potent inhibition of p24 expression in
PBMCs challenged with HIV-1. Overall our results
demonstrate that the lentiviral-based vectors can
efficiently deliver single constructs as well as
combinations of Pol III therapeutic expression
units into primary hematopoietic cells for
anti-HIV gene therapy and hold promise for stem
or T-cell-based gene therapy for HIV-1 infection.
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• Duchenne muscular dystrophy (DMD)
• X-linked recessive disorder
• affects 1 in 3500 live males
• Muscles in patients with DMD are deficient in
  dystrophin, this deficiency causes sarcolemmal
  instability, wich leads to destabilization of the
  sarcolemmal dystrophin-associated protein
  complex.
• low levels of dystrophin are seen in a milder
  form of the disease, the Becker muscular
  dystrophy, BMD.
• Approximately 65 of DMD and BMD patients have
  large deletions in the dystropin gene.

• Dystrophin
• protein 427 KDa
• DNA 2,5 Mb
• cDNA 14 Kb

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AAG
GCA