Gene therapy of neurological diseases Flashcards
Rare diseases
- 7.000–8.000rare diseases are known
- Definition:prevalenceis less than 50/100.000
- Numbers of patients with rare diseases: in Germany 4 Mill., in EU 30 Mill., worldwide 350 Mill.
- Less than 95% of rare diseases can be treated
- 80 % of rare diseases are genetic
How to correct gene defects with gene therapy?
- Expressing of missing protein
- Knockdown of faulty gene (e.g., huntingtin)
- Gene editing by CRISPR/Cas9
In vivo somatic editing therapy
Viral plasmid for editing -> transfection -> packaging cells -> viral production -> viral vectors -> injection -> editing (On and off target analysis)
Vectors for gene therapy
Adeno-associated virus and therapeutic DNA
1. Virus taken into cell via endosome
2. Endosome breaks down
3. Virus binds to cell nucleus and releases contents
4. DNA forms circular episome
Lentivirus and therapeutic RNA EX VIVO
1. Virus taken into cell via endosome
2. Endosome and virus break down releasing RNA
3. RNA is converted into DNA
4. DNA integrates into nuclear genome
5. Protein expressed
6. Corrected cell is reinserted into patient
IN VIVO
Virus is injected directly into patient
Oncolytic virus and therapeutic DNA
1. Virus preferentially infects further cells
2. Virus replicates within cells
3. Tumor cell lyses
4. Immune cells gather to help fight the cancer
Approved gene therapies
-> Alipogen-tiparvovec (Glybera)
- Alipogen-tiparvovec (Glybera®) for the treatment of familial lipoproteinlipase deficiency
Approval: 2012 by EMA - 2018
Costs: 1,100,000 € per patient, only 31 people were treated
Administration: intramuscular
Structure: Capsid from AAV1, CMV promoter, WPRE, AAV2-derived inverted terminal repeats
Approved gene therapies
-> Strimvelis
- Strimvelis® for the treatment of severe combined immunodeficiency (ADA- SCID, ‚bubble disease‘)
Approval: 2016 by EMA
Costs: 594,000 €
Administration: CD34+ stem cells are transduced ex vivo and are reinjected into the patient
Structure: Retrovirus (LXSN)
Approved gene therapies
-> Talimogene laherparepvec (T-VEC, imlygic)
- Talimogene laherparepvec (T- VEC, Imlygic®) for the treatment of metastatic melanomas
- Approval: 2015 by FDA
- Costs: 65.000 $
- Administration: Injection into melanoma lesions
- Structure: Attenuated, replication-competent oncolytic herpes simplex 1 virus producing GM-CSF
Approved gene therapies
-> AstraZeneca vaccine
- Astra Zeneca vaccine
- Spikeproteinis expressed in the replication inefficient chimpanzee adenovirus ChAdOx1
Overview approved gene therapies in the EU
Alipogen Tiparvovec -> Glybera, Unique Biopharma -> Hyperlipoproteinämie Typ I
Talimogen Laherparepvec ->Imlygic, Amgen -> Melanom
autologe CD34 angereicherte Zellfraktion -> Strimvelis, Orchard Therapeutics -> schwerer kombinierte Immundefizieniz (SCID)
Vorrätigen Neparvovec -> Luxturna, Spark therapeutics -> Liberische kongenitale Amouröse
Betibeglogen Autotemcel -> Zynteglo, Bluebird Bio -> Beta-Thalassämie
Onasqmnogen Abeparvovec -> Zolgensma, Novartis -> spinale Muskelatrophie
Atidarsagen Autotemcel -> Libmeldy, Orchard Therapeutics -> metachromatische Leukodystrophie
Elivaldogen Autotemcel -> Skysona, Bluebird Bio -> zerebrale Adrenoleukodystrophie
Eladocagen Exuparvovec -> Upstaza, PTC Therapeutics -> AADC-Mangel
Valoctocogen Roxaparvovec -> Roctavian, Biomarin -> Hämophilie A
Etranacogen Dezaparvovec -> Hemgenix, CSL Behring -> Hämophilie B
Naming of gene therapies
First Word: Corresponds to the Gene Component
Prefix: Fantasy element to provide unique identification; to contribute to the distinct name.
Infix: Element to denote the gene’s mechanism of action (pharmacologic class) such as:
-lip- [human lipoprotein lipase]
–octoco- [coagulation factor Vlll]
-reti- [retinal pigment]
-semn- [SMN]
Stem: Element to indicate gene. -gene
Second Word: Corresponds to the Vector Component
Prefix: Fantasy element to provide unique identification; to contribute to the distinct name
Infix: Element to denote the type of viral vector such as:
-adeno- [adenovirus]
-herpa- [herpes virus]
-lenti- [lentivirus]
-parvo- [adeno-associated virus (parvovirdae dependovirus)]
-retro- [other retro viruses] -vaci- [vaccinia virus]
Stem: Element to identify type of vector
-vec [non-replicating viral vector]
-repvec [replicating viral vector]
-plasmid [plasmid vector]
How to target the CNS?
- Transduction of stem cells ex vivo and infusion of the modified cells
– Problem: slow onset of therapy, risk of leukemia - Intraparenchymal injection of AAV vectors
– Problem: risk of hemorrhage (3%), limited distribution of the vector - High doses of AAV9 to cross the blood-brain barrier
– Problem: off-target effects
Myeloid cells do not infiltrate the brain in healthy adults
- Absence of CNS recruitment in healthy and intact animals
- CNS recruitment following BMT in healthy recipients conditioned by WBI (10 Gy) or toxic chemotherapy regimens
- CNS recruitment following BMT in diseased recipient mice properly conditioned with irradiation or chemotherapy
AAV
- 4700 bases
- 25 nm
- single stranded DNA genome
Production of AAV vectors
- Transfer vector
- pHelper
- pAAV-RC
Subretinal injection
- Bleb (green) between photoreceptors and retinal pigment epithelium
- Transduction of photo receptors
- Compartment is immune privileged
- Technically challenging technique
Voretigene neparvovec (Luxturna)
- ForpatientswithLeber congenital amaurosis (LCA) linked to RPE65
- 5 – 10% of all LCA cases
- Presenting as severe early-onset night blindness
- AAV2-basedexpression of RPE65
Muscle atrophy - muscle dystrophy
Muscle atrophy
* Second most common recessive genetic disease
* Most common genetic cause of death in childhood
* Homozygous loss of SMN1, Phenotype depends on SMN2 copy number
Muscle dystrophy
* Large group of diseases affecting the skeletal muscle and sometimes the heart
* Diverse genetics and phenotypes
Spinal muscle atrophy
Unterschiedliche Formen in absteigender Schwere
- SMA0
- SMA1
- SMA2
- SMA3a
- SMA3b
- SMA4
Onasemnogene abeparvovec (Zolgensma)
- scAAV9 vector with CAG promoter driving ubiquitous SMN1 expression
- Dose: 1.1 x 1014 vg/kg
- Approval: May 2020 by EMA
- Pro:
– One-time intravenous injection
– Systemic distribution - Cons:
– Restricted to individual with < 4 copies of SMN2
– Antibody titer against AAV prevents treatment
– Age limitation due to inability of vector to pass the blood-brain barrier
– Costs 2.1 million US$
Hepatocellular carcinoma due to transduction of hepatocytes?
Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas
Targeting the brain with therapeutic agents
- Chondroitin sulfate targets brain endothelium
- WPFYGTP packaging in the brain blood vessel
- re-entering the brain endothelium
- entering the nucleus
- receptor mediated uptake of the therapeutic protein into the brain
Modification of AAV2-Capsid
-> with insertion of seven random amino acids in the three fold spike domain
Screening of random AAV display peptide libraries in vivo
i.v. injection in mice
-> explantation of tissue of interest
-> isolation of vector DNA from tissue of interest
-> PCR-amplification of library inserts
-> cloning into library plasmids
-> generation of secondary libraries for further rounds of selection by transfection of producer cells
BR1-AAV expression in the central nervous system in vivo -> applications
- Cre-mediated gene recombination in brain endothelial cells
*Incontinentia pigmenti - Cre-mediated gene recombination in brain endothelial cells of Ai14 td-Tomato mice
Incontinentia pigmenti
- Bruno Bloch (1878-1933)
- Marion Baldur Sulzberger (1895-1983)
- Incidence: 1:10.000 - 1:100.000
- Gene: inactivating mutations of NEMO
-Symptoms:
-> start in the neonate
-> skin lesions are diagnostic (Blaschko lines)
-> nails, hair, teeth
-> ocular involvement
-> neurological involvement
Brain lesions determine long-term prognosis
- Spastic paralysis/hemiplegia -> 5.2%
- (psycho)motor delay -> 17.5%
- mental retardation -> 9.4%
- microcepahly -> 3.6%
- seizure disorder -> 12.7%
- cerebellar ataxia -> 0.4%
OVERALL incidence of neurological features -> 31.5%
NF-kappaB signaling
LkappaBalpha + p50 + RelA
-> can lead to lkappaBalpha 2x phosphoryliert
-> can lead to p50 + RelA
String vessel formation in Incontinentia pigmenti mouse
Three day-old female child presented with vesicular eruption and epileptic seizures. She died at 13 days of age after a seizure.
„À l‘autopsie, il n‘y avait pas de malformation cérébrale, mais on observait en coupe CD34 frontale une vaste zone de nécrose sous-corticale … Les vaisseaux étaient normaux.“
Targeted brain endothelial expression of NEMO
- NEMO treatment decreases string vessel formation in neonates
- NEMO treatment improves BBB permeability in neonates
- Delaying seizure onset with BR1-NEMO in NEMObeKO mice
- safety of gene therapy for IP
Enzyme replacement therapy for lysosomal storage diseases
- The endoplasmic reticulum performs the initial processing of newly synthesized lysosomal enzymes
-> Failure of processing in the endoplasmic reticulum can inactivate a whole class of enzymes, such as in multiple sulfates deficiency - The Golgi apparatus further processes and labels specific enzymes for delivery to the lysosome
-> Mutations affecting an enzyme that labels other enzymes for delivery to the lysosome result in mucolipidosis-II - Endosomes transport enzymes from the Golgi and materials from outside the cell to the lysosome
- The autophagosome delivers damaged organelles and misfiled proteins to the lysosome for recycling
-> Defects in a protein that helps endosomes and autpphagosomes fuse with the lysosome cause Danon disease - Within the lysosome, enzymes convert molecules such as sugars, proteins and lipids into simpler building blocks
-> Defects in processing enzymes can cause undigested material to accumulate, such as in GaucherÄ’s disease and Hunter syndrome - Molecular building blocks are released into cell for reuse
-> Loss of transport proteins causes the lysosome to retain molecular building blocs in diseases such as cystinosis or silica acid storage disease
Sandhoff disease
- characterized by the accumulation of GM2 gangliosides
- clinically indistinguishable from Tay-Sachs disease
- clinical heterogeneity
-> infantile manifestation: weakness starts at 6 months, early blindness, progressive mental and motor deterioration, death at the age of 3 years - subacute form: ataxia starting at 2-10 years, mental retardation
- late onset form: motoneuron disease starting in the 3rd decade
beta-hexosaminidase activity after gene transduction
- Gene therapy in Hexb-/- mice
- Gene therapy reduces glial activation in Hexb-/- mice
Gene therapy of neurological diseases - summary
- Genetherapyforcomplexdiseasesofthecentral nervous system is feasible
- Forinvivoapplicationofgenetherapy,mosttrials used AAV-based vectors
- Onasemnogene Abeparvovec (Zolgensma®)has been approved for spinal muscular atrophy
- Administration of vectors to the CNS is possible by direct intraparenchymal injection, vectors that cross the blood-brain barrier (AAV9) or vectors that target the blood-brain barrier (AAV-BR1)
