Gene therapy of neurological diseases

Question 1

Rare diseases

Answer 1

• 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

Question 2

How to correct gene defects with gene therapy?

Answer 2

• 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)

Question 3

Vectors for gene therapy

Answer 3

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

Question 4

Approved gene therapies
-> Alipogen-tiparvovec (Glybera)

Answer 4

• 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

Question 5

Approved gene therapies
-> Strimvelis

Answer 5

• 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)

Question 6

Approved gene therapies
-> Talimogene laherparepvec (T-VEC, imlygic)

Answer 6

• 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

Question 7

Approved gene therapies
-> AstraZeneca vaccine

Answer 7

• Astra Zeneca vaccine
• Spikeproteinis expressed in the replication inefficient chimpanzee adenovirus ChAdOx1

Question 8

Overview approved gene therapies in the EU

Answer 8

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

Question 9

Naming of gene therapies

Answer 9

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]

Question 10

How to target the CNS?

Answer 10

• 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

Question 11

Myeloid cells do not infiltrate the brain in healthy adults

Answer 11

• 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

Question 12

AAV

Answer 12

• 4700 bases
• 25 nm
• single stranded DNA genome

Question 13

Production of AAV vectors

Answer 13

1. Transfer vector
2. pHelper
3. pAAV-RC

Question 14

Subretinal injection

Answer 14

• Bleb (green) between photoreceptors and retinal pigment epithelium
• Transduction of photo receptors
• Compartment is immune privileged
• Technically challenging technique

Question 15

Voretigene neparvovec (Luxturna)

Answer 15

• ForpatientswithLeber congenital amaurosis (LCA) linked to RPE65
• 5 – 10% of all LCA cases
• Presenting as severe early-onset night blindness
• AAV2-basedexpression of RPE65

Question 16

Muscle atrophy - muscle dystrophy

Answer 16

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

Question 17

Spinal muscle atrophy

Answer 17

Unterschiedliche Formen in absteigender Schwere
- SMA0
- SMA1
- SMA2
- SMA3a
- SMA3b
- SMA4

Question 18

Onasemnogene abeparvovec (Zolgensma)

Answer 18

• 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$

Question 19

Hepatocellular carcinoma due to transduction of hepatocytes?

Answer 19

Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas

Question 20

Targeting the brain with therapeutic agents

Answer 20

1. Chondroitin sulfate targets brain endothelium
2. WPFYGTP packaging in the brain blood vessel
3. re-entering the brain endothelium
4. entering the nucleus
5. receptor mediated uptake of the therapeutic protein into the brain

Question 21

Modification of AAV2-Capsid

Answer 21

-> with insertion of seven random amino acids in the three fold spike domain

Question 22

Screening of random AAV display peptide libraries in vivo

Answer 22

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

Question 23

BR1-AAV expression in the central nervous system in vivo -> applications

Answer 23

• Cre-mediated gene recombination in brain endothelial cells
  *Incontinentia pigmenti
• Cre-mediated gene recombination in brain endothelial cells of Ai14 td-Tomato mice

Question 24

Incontinentia pigmenti

Answer 24

• 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

Question 25

Brain lesions determine long-term prognosis

Answer 25

• 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%

Question 26

NF-kappaB signaling

Answer 26

LkappaBalpha + p50 + RelA
-> can lead to lkappaBalpha 2x phosphoryliert
-> can lead to p50 + RelA

Question 27

String vessel formation in Incontinentia pigmenti mouse

Answer 27

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.“

Question 28

Targeted brain endothelial expression of NEMO

Answer 28

• 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

Question 29

Enzyme replacement therapy for lysosomal storage diseases

Answer 29

1. 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
2. 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
3. Endosomes transport enzymes from the Golgi and materials from outside the cell to the lysosome
4. 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
5. 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
6. 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

Question 30

Sandhoff disease

Answer 30

• 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

Question 31

beta-hexosaminidase activity after gene transduction

Answer 31

• Gene therapy in Hexb-/- mice
• Gene therapy reduces glial activation in Hexb-/- mice

Question 32

Gene therapy of neurological diseases - summary

Answer 32

• 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)