1. PERSPECTIVAS EN
TERAPIA DE
ENFERMEDADES
GENÉTICAS
Dra. Ana Prötzel Pinedo

2. 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.

6. Capacidades adquiridas para el desarrollo de
cáncer

7. 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 transcripción
Reguladores del ciclo celular
 At surrounding/environment level
Factores neoangiogénicos
Matrix Metalloproteinases
 A nivel de organismo:
Evasión del Sistema inmune

8. Posibilidades Técnicas:
Diseño computacional de nuevas drogas:
Inhibidores químicos
Peptidomiméticos
Anticuerpos
Oligonucleótidos
Immunoterapia: Uso de anticuerpos
Potenciación del sistema inmune
Gen terapia: Células asesinas por bioactivación de
prodrogas o por replicación selectiva de
virus.
Reintroducción de genes supresores de
tumor o silenciamiento de oncogenes.
Potenciación del sistema inmune.

9. Areas en el desarrollo de la Terapia Génica
Vectores

12. Areas en el desarrollo de la gen terapia:
Vectores
Altos títulos y fácil preparación,
Adenovirus amplio trofismo, especialmente
bueno para células epiteliales
Altamente inmunogénicas
Virales Retrovirus Bajos títulos, dependencia de
células en división, útil in vitro.
Parvovirus Lentivirus (HIV) no requiere
células en división.
Otros: herpes virus Integrativos, pequeños.
H1 es oncotrópico.
reovirus, alphavirus..
Complejos
DNA DNA desnudo, Plásmidos, ...
Liposomas y complejos
policatiónicos.Drogas clásicas
Liposomas (Doxil). Menos toxicidad y menos
eficacia.

15. Estrategias de administración

16. Estrategias de Administración

17. Vector no Vector
dirigido
dirigido
(“targeted”)

18. 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.

19. Viroterapia génica:

20. Areas en el desarrollo de terapia génica
Genes

22. Dos áreas en el desarrollo de terapia
Genes Vectores

23. Prodrug bioactivators genes/suicide genes
SUICIDE
Non toxic Toxic drug
Prodrug ENZYME
1. Introduction 2. Prodrug 3. Cell death
of suicide gene Administration
+

25. Prodrug bioactivators genes: bystander effect
suicide enzyme
Toxic
prodrug metabolite
prodrug
+

27. Tumour suppresor and oncogenes

28. Oligonucleotidos antisentido en ensayos clínicos
Compound Target Indications Devel. phase
 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
Baker & Monia, 1999

29. RNAi: Molecular understanding

31. Gene silencing mediated by siRNAs/shRNAs
19 nt duplex
5’ 3’ 19-21 nt duplex
GGG A AG
3’ 5’
2nt 2nt UUUUUCCC C
U U
overhang overhang
Synthesized oligonucleotides SiRNA Hairpin RNA
mRNA
Antisense strand RNAi silencing
Complex
mRNA cleavage
Degraded mRNA

33. 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.

34. "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).

35. 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.

37. Gracias…..