PERSPECTIVAS EN

TERAPIA DE

ENFERMEDADES

GENÉTICAS

Dra. Ana Prötzel Pinedo

1960: Primeras evidencias de falta de expresión del DNA exógeno en células de

mamíferos

Discusión de políticas públicas, aspectos éticos y potenciales aplicaciones en

humanos

1970: Mecanismos de infección y transcripción reversa por virus tumorales RNA

Estudios clínicos en humanos (Israel, 3 pacientes)

Se reanudan las discusiones sobre políticas públicas.

1989-1990: Se aprueban los primeros estudios clínicos y potenciales terapias

TILs amb RV-Neo

T-cells y RV-ADA

Plásmido-B7.1 en melanoma

RV-Ras-antisentido en NSCLC (Cancer pulmonar a células no pequeñas)

1999: Muere Jesse Gelsinger (18a) quién participaba en un estudio clínico para

terapia génica para OTCD. Murió por falla orgánica multisistémica, 4 días

después de iniciar el tratamiento.

2001: El programa de SCID (Inmunodeficiencia combinada severa) en Francia y

UK. En 2002, dos niños enrolados en el programa francés desarrollaron

Leucemia.

2003: Se logra atravezar la barrera H-E con liposomas.

Potencial tratamiento para la Enfermedad de Parkinson

2006: Uso de miRNAs en el rechazo del sistema inmune a los genes recién

introducidos.

2007: Primera terapia génica administrada para enfermedad retinal hereditaria.

Capacidades adquiridas para el desarrollo de

cáncer

Estrategias en Células de cáncer

A nivel de membrana plasmática:

Receptores de Factor de crecimiento

Integrinas

Moléculas de adhesión Cadherinas

Conexinas

A nivel intracelular:Vias de señalización

Factores de transcripciónReguladores del ciclo celular

At surrounding/environment level

Factores neoangiogénicos

Matrix Metalloproteinases

A nivel de organismo:

Evasión del Sistema inmune

Posibilidades Técnicas:

Diseño computacional de nuevas drogas: Inhibidores químicosPeptidomiméticosAnticuerposOligonucleótidos

Immunoterapia: Uso de anticuerposPotenciación del sistema inmune

Gen terapia: Células asesinas por bioactivación deprodrogas o por replicación selectiva devirus.Reintroducción de genes supresores detumor o silenciamiento de oncogenes.Potenciación del sistema inmune.

Areas en el desarrollo de la Terapia Génica

Vectores

Areas en el desarrollo de la gen terapia:

Vectores

Virales Retrovirus

Parvovirus

AdenovirusAltos títulos y fácil preparación, amplio trofismo, especialmente bueno para células epiteliales Altamente inmunogénicas

Bajos títulos, dependencia de células en división, útil in vitro.

Lentivirus (HIV) no requiere células en división.

Integrativos, pequeños.H1 es oncotrópico.

DNA desnudo, Plásmidos, ...

Liposomas y complejos policatiónicos.Drogas clásicas (Doxil). Menos toxicidad y menos eficacia.

Otros: herpes virusreovirus, alphavirus..

Complejos DNA

Liposomas

Estrategias de administración

Estrategias de Administración

Vector no

dirigido

Vector

dirigido

(“targeted”)

Pruebas clínicas usando vectores

retrovirales:

Enfermedades genéticas (inmunodeficiencias primarias):

Deficiencia de ADA : Deficiencia de Adenosin D aminasa

Primer desórden tratado con gen terapia en dos pacientes (1990)

Se usó Rv-transducidos a células T de linfocitos de sangre periférica.

Tratamiento concomitante con PEG-ADA, 10 años después de la última

infusion todavía hay expresión.

Transplante de MO CD34+ HSC transductas, produjo restauración de la

función inmune, corrección del defecto de ADA y beneficio clínico.

X-SCID: (2000) Primera gen terapia exitosa.

(2002 –2005) 3/14 pacientes, desarrollaron leucemia.

X-CGD( X-linked chronic granulomatous disease):

(2006) Defecto en la actividad antimicrobiana oxidativa de los fagocitos . Se utiliza MO CD34+ HSC transductas y Gamma-retrovirus en 2 pacientes. Se logra corrección funcional.

Viroterapia génica:

Areas en el desarrollo de terapia génica

Genes

Dos áreas en el desarrollo de terapia

Genes Vectores

Prodrug bioactivators genes/suicide genes

Non toxic

Prodrug

Toxic drugSUICIDE

ENZYME

1. Introduction

of suicide gene3. Cell death2. Prodrug

Administration

+

Prodrug bioactivators genes: bystander effect

suicide enzyme

+

prodrug

prodrug

Toxic

metabolite

Tumour suppresor and oncogenes

Oligonucleotidos antisentido en ensayos clínicos

Vitravene (ISIS 2922)CMV IE2 gene CMV retinitis Approved

ISIS 2302 ICAM-1 Crohn’s disease Phase III

ISIS 3521 PKC-a cancer Phase II

ISIS 5132 C-raf kinase cancer Phase II

G 3139 Bcl-2 cancer Phase II

INX 3280 c-myc restenosis Phase II

ISIS 2503 Ha-ras cancer Phase II

GEM 132 CMV UL36 geneCMV retinitis Phase I

ISIS 13312 CMV IE2 gene CMV retinitis Phase I

GEM 91 DNA metiltransferas cancer Phase I

Compound Target Indications Devel. phase

Baker & Monia, 1999

RNAi: Molecular understanding

Gene silencing mediated by siRNAs/shRNAs

5’ 3’

3’ 5’2nt 2nt

overhang overhang

19 nt duplex

SiRNA

mRNA

RNAi silencing Complex

Degraded mRNA

Antisense strand

19-21 nt duplex

UUUUUCCCGGG A A

GC

UU

Synthesized oligonucleotides Hairpin RNA

mRNA cleavage

The nanoparticles are extremely small, ranging from 185 to 375 nanometers (a nanometer is one billionth of a meter, or a millionth

of a millimeter). For comparison, red blood cells are ten to 100 times larger. The researchers were able to control the nanoparticle

size by varying the amount or composition of solvents they used to form the nanoparticles.

The magnetically driven delivery system also may find broader use as a vehicle for delivering drugs, genes or cells to a target

organ. This is a novel delivery system, the first to use a biodegradable, magnetically driven polymer to achieve clinically relevant

effects.

Impregnated with iron oxide, the nanoparticles carry a surface coating of DNA bound to an organic compound called

polyethylenimine (PEI). The PEI protected the DNA from being broken down by enzymes called endonucleases that were present

in the cell cultures and which occur normally in the bloodstream.

The DNA was in the form of a plasmid, a circular molecule that here carried a gene that coded for a growth-inhibiting protein called

adiponectin. By applying a magnetic field, the study team steered the particles into arterial smooth muscle cells. Inside each cell,

the DNA separated from the particle, entered the cell nucleus, and produced enough adiponectin to significantly reduce the

proliferation of new cells.

The materials composing the nanoparticles are biodegradable, so they break down into simpler, nontoxic chemicals that can be

carried away in the blood. "Previous researchers had shown that magnetically driven nanoparticles could deliver DNA in cell

cultures, but ours is the first delivery system that is biodegradable, and therefore, safer to use in people," said Levy.

"Fifty million Americans suffer from chronic pain. Chronic pain patients

often do not experience satisfactory pain relief from available

treatments due to poor efficacy or intolerable side effects like extreme

sleepiness, mental clouding, and hallucinations,"

Mount Sinai researchers designed a viral vector to carry the prepro-b-

endorphin gene into primary sensory neurons in order to activate

opiate receptors selectively, in a rat model. The agents were delivered

directly into the spinal fluid of rats via a lumbar puncture, or spinal tap

with only one injection. Results showed that the rats remained

symptom-free for an extended period of time.

"Our research found that treating chronic pain with Adeno-Associated

Virus vector-based gene therapy allows for pain relief for more than

three months after a single injection, targeting selectively the pain

gate.

Based on these findings, this targeted gene therapy via lumbar

puncture appears to be a promising candidate for bench-to-bedside

research that might ultimately be tested in patients with intractable

chronic pain, e.g., to help patients suffering from severe pain due to

advanced cancer.“

The study "Sensory neuron targeting by self-complementary AAV8 via

lumbar puncture for chronic pain" was published in the January 22,

2008 issue of the Proceedings of the National Academy of Sciences

(PNAS).

Eight patients were enrolled on the trial and six were infused

with their own stem cells which were engineered to carry the

MGMT (O6-methylguanine–DNA methyltransferase) gene. In

three patients, stem cells carrying the gene were identified in

their blood or bone marrow. In one patient, stem cells carrying

the gene were detected up to 28 weeks after their

administration. This significant finding has never been reported

before with this gene and drug combination.

'This study is the first to show the success of treatment with

evidence that stem cells now carry the new gene,'¨ says Dr.

Gerson, Director of the Ireland Cancer Center and Case

Comprehensive Cancer Center, who spearheaded the Phase I

study along with a team of researchers. 'These patients show

the success of treatment with evidence that their stem cells now

carry the new genes. This is a breakthrough -- the first time

selection with MGMT has been shown to occur in patients.'¨

Preclinical animal research, conducted by Dr. Gerson and his

colleagues, has shown that the gene G156A-MGMT can provide

stem cells with very high levels of drug resistance, compared to

normal stem cells not carrying the gene. In the Phase I trial for

patients with advanced malignancies, researchers collected

peripheral blood stem cells from patients and exposed them to a

retrovirus containing the G156A-MGMT gene.

Gracias…..