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