adn, arn, y proteína · adn, arn, y proteína. arn versus adn. expresión génica. el código...
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ADN, ARN, y proteína
ARN versus ADN
Expresión génica
El código genético
Estructura de un gen y pasos de la expresión génica que ocurren en el núcleo
ATG TAA
poly(A)TSS
transcription
exonintron
AAAAA
3’-end processing
5’UTR 3’UTR
splicing
AAAAA
Datos de interés
• ~100 billones de células en el cuerpo humano
• 2 × 3.000 millones de pares de bases de ADN en cada célula
• ~25,000 genes en cada célula
• El gen promedio tiene 27,000 pares de bases y 8 intrones
• ~200,000 intrones en cada célula
Pre-mRNA splicing
exon intron
5’ splice site
3’ splice site
exonPre-mRNA
mRNA
the spliceosome(~170 proteins + 5 snRNAs)
El spliceosoma corta y empalma con gran precisión>200,000 uniones a lo largo de ~25,000 cables!
Atrofia Muscular Espinal
• Principal causa genéticade mortalidad infantilde mortalidad infantil
•Degeneramiento progresivo de las motoneuronas
• Por ahora no existe cura
Spinal Muscular Atrophy
• Pediatric neuromuscular disorder
• Degeneration of motor neurons in the spinal cord
• Incidence: 1 in ~6,000 live births
• Autosomal recessive: child of 2 carriers has 1 in
4 chance of being affected
• Variable severity (type I, II, III, IV)
• Mutant or missing SMN1 gene
• Related SMN2 gene provides partial function, due
to defective splicing
Inverted duplicationCentromeric Telomeric
HRAD17 p44c ψ NAIP SMN2 H4F5c H4F5t SMN1 NAIP p44t TFNR
q11.2 q34q14.3
Survival of motor neuron 1, telomeric (SMN1)es el gen mutado en la AME
q13
Chr. 5
5q13
A2G D30N D44V G95R
V94G
W92S A111G
I116F
Y13 0C
E134K
Q136E
A188S
P245LL260S
M263R/T
S262I/G
S266P Y272C
G279V/C
G275S
T274I
H273R
Dimerization Dimerization
Tudor YGP-rich
Gemin2binding Sm binding
K-rich
Survival Motor Neuron (SMN) protein Adapted from Rossoll & Bassell (2009) Results Probl Cell Differ 48: 289
1 2a 2b 3 4 5 6 AAAAAA87
SMN1 gene
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8SMN1 mRNA
Splicing de SMN1 y SMN2
11 22 33 44 55 66 77 88
SMN2 gene
1 2 3 4 5 6 8SMN2 mRNA
8
Un peque ňo cambio en la secuencia hace que se saltee el exon 7 de SMN2
STOP
AAAA7SMN1 6~100%
SMN
8
STOP
AAAA
GGT TTT AGA
GGT TTC AGA
SMN2 7650 - 90%
10 - 50%
SMN∆∆∆∆7
ADN (gen) mRNA
Moléculas antisense
transcriptionA - T(U)G - C
ASO
8U 76 876
Método para aumentar la inclusión del exon 7
868U 76R
2'-O-(2-methoxyethyl) ribose
phosphorothioate
5-methyl cytosine
G
OCH2CH2OCH3
O
A
OCH2CH2OCH3
O
T
OCH2CH2OCH3
O
C
OCH2CH2OCH3
O
P
O
O
OS
P
O
O
OS
P
O
O
OS
P
O
O
OS
O
Yimin Hua
ISS-N1
GUAAGUCUGCCAGCAUUAUGAAAGUGAAUCUUACUUUUGUAAAACUUUAUGGUUUGUGGA …
U1
ISE I7-1
A1/A2 A1/A2
ISIS-SMNRx (ASO-10-27)
Exon 7
ISIS-SMNRx: nuevo fármaco en investigación
ISIS-SMNRx
18mer; MW 7127Hua et al (2007) PLoS Biol 5: e73Hua et al (2008) Am J Hum Genet 82: 834Hua et al (2010) Genes Dev 24: 1634Passini et al (2011) Science Transl Med 3: 72ra18Hua et al (2011) Nature 478: 123
Pre-clinical development
GMPmanufacture
Targetdiscovery
Hit IDLead
optimizationSafety &
tolerability
Descubrimiento y desarrollo de fármacos
ApprovalPhase
IIPhase
IIIPhase
IINDfiling
Clinical testing
Modelo de AME en ratón
Hsieh-Li et al (2000) Nature Genet 24: 66-70
Monani et al (2000) Hum Mol Genet 9: 333-339
Motoneuronas- α en la espina dorsal
14 8614 13 84 83
Saline ASO
% incl
RT-PCR
Rescue by neonatal ICV injection of ASO
% s
urvi
val
20
40
60
80
100 ICV-SalineICV-ASO6 7 8
6 8
Dose: 20 µg ICV at P1Tissue: whole spinal cord at P7
Mean life span
Saline: 10 daysASO: 17 days
0 5 10 15 20 25 30
Age (days)
% s
urvi
val
20
0
Saline ASO
hSMN
Tubulin
Western
Intrathecal drug delivery
Intrathecal drug delivery
Current status of ISIS SMN Rx
• Phase I clinical trial started in December 2011
• Open-label safety, tolerability and dose-range finding study
• Single intrathecal injection at one of four different doses• 24 patients (four dose levels, six patients each)
• Eligibility: 2 to 14 year old males and females, >2 year life expectancyexpectancy
• Locations:– Columbia University Medical Center, New York City
– University of Utah School of Medicine, Salt Lake City
– Children’s Hospital, Boston
– UT Southwestern Medical Center, Dallas
• Jan 4: Biogen-Idec partnership• Phase Ib/2a multiple-dosing trial just announced
Dose-dependent survival after SC injections
Sur
viva
l (%
)
Heterozygotes
60
80
100
Age (days)
Sur
viva
l (%
)
Saline
160 µg/g80 µg/g40 µg/g
2 SC injections at P1 & P3
0 150 300 450 600 7500
20
40
Smn-/- ; SMN22Tg/0Hua et al (2011) Nature 478: 123
α-motor neuron counts
10
15
20
25
mot
or n
euro
ns
P < 0.01
SMA + saline
Sections from L1-L2 at P92 × 80 µg/g SC ASO injections at P1 & P3
0
5
10
Het SMA+ASO SMA-saline#
of α
-m
ChAT staining
DAPI
SMA + ASO
Rotarod performance test
P90
2 ×160 µg/g SC ASO injections at P1 & P3 Untreated heterozygote control
Territorial behavior: SC160 versus heterozygotes
P90
2 ×160 µg/g SC ASO injections at P1 & P3 Untreated heterozygote controls
Yimin HuaXavier RocaHazeem Okunola
Krainer Lab 2012
Mads Jensen
Akila MayedaMichael Murray
Hong-Xiang LiuEric Allemand
Javier Cáceres
Shern Chew
Ewan Birney
Luca Cartegni
Former members
Isabel AznárezOlga Anczuków
Deblina ChatterjeeShipra Das
Martín Akerman
Fatma Bezirci
Rahul Sinha
Zuo Zhang
Xavier Roca
Lisa MancheYing Shiu Liu
Mads Jensen
Shuying Sun
Qing-Shuo ZhangQiang Wu
Jun Zhu
Tzu-Ling TsengIkuko Watakabe
Diane Kozak
Michelle Hastings
Benhao DongShern Chew
Akemi Hanamura
Geeta Joshi-TopeDavid Horowitz
Philip Smith
Rotem KarniBernard Khoo
Stephanie Shaw
Ruei-Ying Tzeng
Oliver Fregoso
Kentaro Sahashi
Zhenxun Wang
Michelle Faleiro
Jaclyn Novatt
Lisandra Zepeda
Kwan-Ting LinHyun-Yong Jeon
Yilei Liu
Collaborators and SMA funding
Frank BennettFrank Rigo Gene Hung
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