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1. For more extensive expression:
To improve our vector, AAV8 could be a choice.
Considering multiple injection sites for a repeated vector dose, this might have better performance than the original single shot.

2. Later effects:
After administration of the drug, long-term effects still need to be monitored. Also, we may need to investigate the potential toxicities of our gene therapy.

3. May keen more on mechanism:
The spreading depolarization on the hippocampus causes the symptoms of AHC. Next time we should try to alter the physiology.

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Hope for Alternating Hemiplegia of Childhood

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How AAV Gene Transfer Works - General Audience 

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- AAV genetic material is replaced with a therapeutic gene to restore natural function of the disrupted protein
- protein shell called capsid encapsulates the genetic material to deliver to target cells
- in target cells, capsid is shed to release genetic material
- genetic material produces episomes (not passed onto germ line cells)
- allows stable and long term production of therapeutic molecule!

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How AAV Gene Transfer Works - General Audience

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For the PowerPoint presentation:

Zunaynah Shuha - Introduction, Experiments IIIA, IIIB and IV, slides - Points of discussion and Bibliography

Tahiyat Islam - Methods, Experiments I and II

Runze Wang - Discussion

For the padlet ~ each member contributed the post they are the author of

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Bibliography (used for the introduction, experiments IIIA, IIIB & IV)

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Bibliography (used for the introduction, experiments IIIA, IIIB & IV)

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1. Hunanyan, A. S., Kantor, B., Puranam, R. S., Elliott, C., McCall, A., Dhindsa, J., Pagadala, P., Wallace, K., Poe, J., Gunduz, T., Asokan, A., Koeberl, D. D., ElMallah, M. K., & Mikati, M. A. (2021). Adeno-associated virus-mediated gene therapy in the mashlool, Atp1a3Mashl/+, mouse model of alternating hemiplegia of Childhood. Human Gene Therapy, 32(7–8), 405–419.

2. https://www.tiktok.com/@jordandaniellesil?is_from_webapp=v1&is_copy_url=1&item_id=6953479054976748805

3. Salles, P. A., Mata, I. F., Brünger, T., Lal, D., & Fernandez, H. H. (2021). ATP1A3-related disorders: An ever-expanding clinical spectrum. Frontiers in Neurology, 12, 637890.

4. Samanta, D. (2020). Management of alternating hemiplegia of childhood: A review. Pediatric Neurology, 103, 12–20.

5. About — Cure AHC. (n.d.). Cureahc.Org. Retrieved November 12, 2021, from https://www.cureahc.org/about

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Overlapping of ATP1A3-related disorders. (Salles et al., 2021)

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Proposed model can work for syndromes under the umbrella of ATP1A3-Related Spectrum Disorders

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Proposed model can work for syndromes under the umbrella of ATP1A3-Related Spectrum Disorders

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ATP1A3-related ATPase dysfunction ³, a newly recognized mechanism for neuronal injury, in -

disorders manifesting energy crises e.g. status epilepticus or hypoxia

neurodegenerative disorders e.g. Parkinson’s and Alzheimer’s.

disorders with secondary ATP1A3-related ATPase dysfunction.

Proposed model can be used in the therapy of deficiency diseases of other ATPases, e.g. V-ATPases, which are important in multiple types of human disease¹.

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Both figures from Hunanyan et al., 2021. Color change of Figure B created with BioRender.com

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Experiment IV: Quantification of test vector transgene expression by western blot analyses of mice organs at P40 using FLAG-tag antibody after completion of the behavioral tests

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Experiment IV: Quantification of test vector transgene expression by western blot analyses of mice organs at P40 using FLAG-tag antibody after completion of the behavioral tests

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Western blot showed adult mice injected at P10 expressed FLAG - indicating continued expression of the transgene into adulthood, although more so in the hippocampus than in the cerebellum¹.

Results indicate efficient vector distribution and transduction into brain areas and the expected concurrent, lower transduction into studied organs.

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Figure A created with BioRender.com. Survival curve of Figure B from Hunanyan et al., 2021. 

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Experiment IIIB: effects of optimized test vector on phenotype during adulthood - injected mice allowed to mature into full adulthood and behavioral tests repeated on surviving mice at P70.

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Experiment IIIB: effects of optimized test vector on phenotype during adulthood - injected mice allowed to mature into full adulthood and behavioral tests repeated on surviving mice at P70.

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Number of hindlimb slips on the balance beam test in the Het-treatment group significantly lower than in the Het-control group¹.

As seen in the survival curve, significant reduction in mortality of Hets that received treatment compared to Het-controls observed.

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Proposed model AAV-directed delivery of a functional copy of the ATP1A3 gene. Created with BioRender.com

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Diagnosis⁴ - onset of symptoms before reaching 18 m/o.

Available drugs only alleviate the more severe symptoms ³ e.g. seizures.

Presently, there is no cure for AHC ¹⁻⁵.

As a severe orphan disease - it qualifies for the special facilitated FDA programs for orphan diseases¹.

Proof of concept study¹: authors demonstrated AAV-directed delivery of a functional copy of the ATP1A3 gene ameliorates some of the manifestations of AHC in the mouse model expressing the D801N mutation.

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De novo mutations 4,5 in the ATP1A3 gene contributing to approximately 80% of AHC cases.

Most common D801N (43%) mutation confers a milder phenotypic expression

E815K (25%) mutation is associated with a more severe phenotype-more severe intellectual and motor disability.

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FLAG-TAG antibody staining (seen as green fluorescence), mCherry (red) and DAPI (blue).  Green areas = where transgene is being expressed.

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Picture A & B shows the hippocampal CA3 region expression (this had unilateral ICV injection of a high dose (20 x 10^10 vg)

Picture C & D are of the hippocampal CA1 region expression after injection of the ICV dose

From picture A-D, you see robust expression in the ipsi lateral hippocampus (+3 score for both mCherry and FLAG-tag)

In figure 2F, we examine cerebellar reporter gene expression after ICM injection |  +3 score of mCherry and flag

In figure 2I to 2k, we see that the cortex and cerebellum showed an overall expression score of +1 but higher expression scores of +3 in areas close to the injection sites

Lower expression scores  of 0 or +1 were seen in more distal regions, as can be seen in figure 2L- 2N;

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Some early symptoms²:

irregular eye movements

difficulty swallowing

involuntary limb movements

paralysis

Shared symptoms with other diseases 4,5:

movement problems like Cerebral Palsy

learning challenges like autism

behavioral issues like ADHD

true epilepsy in at least 50% of cases

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@jordandaniellesil
Reply to @jackelineeg Early symptoms! #alternatinghemiplegiaofchildhood #AHC #ahcawareness #1inamillion
♬ You Are My Sunshine - Kina Grannis

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Alternating Hemiplegia of Childhood (AHC)

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Alternating Hemiplegia of Childhood (AHC)

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A rare neurological disorder ⁴ characterized by repetitive episodes of paralysis.

Paralysis can affect one or both sides of the body, multiple limbs or a single limb.

The bouts of paralysis can last from a couple of minutes to a few days.

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@jordandaniellesil
Alice's story... #alternatinghemiplegiaofchildhood #AHC #1inamillion #donate
♬ You Are The Reason - Alex Porat

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- there is an optimal dose at which robust transgene expression is achieved --> after combined injections (bilateral ICV = ICM) they found robust expression of both markers, particularly of FLAG with the higher dose of 27 x 10^10 vg per mouse
- hippocampus showed FLAG expression score of +3 in CA3 and CA1
- 0 to +1 in areas that are more distal
- ATP1A3-FLAG fusion protein was detected in the cell membrane → this tells us that the virally expressed ATP1A3 transgene is expressed but also that the ATP1A3 protein is transported to its functional site, the cell membrane

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- how injection of the test vector affects transgene expression and also what dose of the test vector is the optimal dose?
- injected ICM and bilateral ICV injections at the same time in each mouse; different doses
- three groups of mice, each receiving a varying dose of the vector

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0 = no expression

+1 = 1-25% of cells expressing transgene

+3 - 50-100% cells expressing transgene

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Experiment IIIA: optimized test vector dosing improved balance beam performance at P40

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Experiment IIIA: optimized test vector dosing improved balance beam performance at P40

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Significantly shorter balance beam test time of the Het-treatment group compared to the Het-control group¹.

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Experiment IIIA: behavioral tests and hemiplegia spell frequency compared between 5 groups

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Experiment IIIA: behavioral tests and hemiplegia spell frequency compared between 5 groups

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To determine the effects of optimized test vector obtained from experiment II on phenotype during pubescence¹ - compared five groups of animals at P40 after injecting the mice at P10.

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1. The choice of mouse model:
By conferring the common mutation of the AHC, the mice model honestly expressed the situation of the disease. And the age of mice was parallel to human age during the experiment, which was strict.

2. The choice of promoter:
hSyn is a neuron-specific promotor, ensuring transgene expressions happen in neurons instead of glial cells.

3. The choice of virus:
AAV has good CNS transduction. which promotes the potential of translation. And it is quite safe for therapeutic usage (won't trigger a fierce immune respense}

4. No worsening for Het-treatment group compare to the control:
This phenomenon is fascinating and proved harmlessness of adding an extra copy of the gene.

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Het-treatment mouse vs Het-control mouse displaying paralysis of hind legs. Created with BioRender.com

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Experiment IIIA: optimized test vector dosing improved hemiplegia spell occurrences at P40

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Experiment IIIA: optimized test vector dosing improved hemiplegia spell occurrences at P40

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Significantly reduced occurrence of hemiplegic spells in the Het-treatment group compared to the Het-control group¹ - hemiplegia occurred in 50% of Het-control animals and in none of the Het-treatment mice.

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A piece of solid evidence supported the 2nd explanation. By tracing FLAG-tag in the mouse brain among different periods, the authors found far more FLAG in P40 than P70 (4 times more), which means there are more transgene expressions in the early stage(FLAG is inside the vector gene), vector effect loss by age.

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Main conclusion: By adding copies of ATP1A3, the performance of mice in multiple tests was improved, indicating the amelioration of AHC disease.

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If, during P40, the Het-CTRL group (except for dead mice) showed no difference on the balance beam task compared with the Het-treatment group, the 1st hypothesis might be the right one.

The analysis result showed a difference, disagree with the 1 st hypothesis

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During P40, the Het-treatment group did better in the performance test than the control group, stating a better AHC condition. But this advantage was not appearing in adulthood. Why would this happen?

Possible reasons:

1. Het-control with lethal situation died after P40, so comparing the leftover with treatment group might make a biased result

2. The effect of the viral vector loss by age, but the AHC related neurodegeneration is still ongoing. By the way, AAV9 cannot propagate into newborn neurons, while children still have mass neurogenesis.

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- test vector increased ATPase activity
- performed and analyzed ATPase enzyme activity assays
- test vector group’s ATPase activity to be 102% higher in the ipsilateral hippocampus than in hippocampus of the control vector group --> similar results seen in cerebral cortex, basal ganglia/thalamus, and brain stem

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- how does the test vector affect ATPase enzyme activity?
Compared two groups of WT mice
1. injected an active vector in the right lateral ventricle at P10--> sacrificed at P40
2. injected with a control vector at p10 and killed at p40.

compared the ATPase enzyme activity in the right hippocampus and in other ipsilateral brain regions (cortex, thalamus, basal ganglia, brainstem, and cerebellum) of both groups.

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- D801N mutant mice
- use AAV9 vectors to deliver wt ATP1A3 gene
- FLAG-tag sequence flanking the transgene
- control vector: AAV with no transgene

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Diagram of test vector, containing the transgene as well as the FLAG-tag sequence and mCherry red fluorescent tag.

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- mice were injected at 10 days old (P10) --> correspond to the early infancy period in humans
- behavioural testing, following AAV injection at P40