USING AAV GENE REPLACEMENT THERAPY TO TREAT ALTERNATING HEMIPLEGIA OF CHILDHOOD (AHC) 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. by Tahiyat Islam https://padlet.com/tahiyat01/14oa56742y6amy1w Made with a creative frenzy en-us 2021-12-07 01:39:13 UTC 2025-11-13 05:06:30 UTC hello@padlet.com METHODS tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932482313 - D801N mutant mice
- use AAV9 vectors to deliver wt ATP1A3 gene
- FLAG-tag sequence flanking the transgene
- control vector: AAV with no transgene]]> 2021-12-07 01:41:22 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932482313 tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932494603 2021-12-07 01:49:16 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932494603 Now...let's inject! tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932495337 - mice were injected at 10 days old (P10) --> correspond to the early infancy period in humans
- behavioural testing, following AAV injection at P40]]> 2021-12-07 01:49:45 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932495337 tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932506583 2021-12-07 01:57:04 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932506583 EXPERIMENT I: AIMS tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932507931 - 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.]]> 2021-12-07 01:57:54 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932507931 EXPERIMENT I: RESULTS tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932508407 - 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]]> 2021-12-07 01:58:11 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932508407 Not working in adult? runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520027 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.]]> 2021-12-07 02:05:40 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520027 Verification runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520487 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]]> 2021-12-07 02:05:59 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520487 AHC amelioration runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520633 Main conclusion: By adding copies of ATP1A3, the performance of mice in multiple tests was improved, indicating the amelioration of AHC disease.]]> 2021-12-07 02:06:05 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520633 Discussion runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520748 2021-12-07 02:06:10 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932520748 Vector effect loss by age runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932585062 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.]]> 2021-12-07 02:45:19 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932585062 Experiment IIIA: optimized test vector dosing improved hemiplegia spell occurrences at P40 zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932592374 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.]]> 2021-12-07 02:49:54 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932592374 Strength runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932614288 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.]]> 2021-12-07 03:04:12 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932614288 Experiment IIIA: behavioral tests and hemiplegia spell frequency compared between 5 groups zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932619074 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.]]> 2021-12-07 03:07:25 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932619074 Experiment IIIA: optimized test vector dosing improved balance beam performance at P40 zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932619241 Significantly shorter balance beam test time of the Het-treatment group compared to the Het-control group1.]]> 2021-12-07 03:07:33 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932619241 Levels of Gene Expression: tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633005
  • 0 = no expression
  • +1 = 1-25% of cells expressing transgene
  • +3 - 50-100% cells expressing transgene
    • ]]>     2021-12-07 03:16:59 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633005     EXPERIMENT II: AIMS tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633162 - 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]]>     2021-12-07 03:17:05 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633162     EXPERIMENT II RESULTS: tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633304 - 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]]>     2021-12-07 03:17:10 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633304     Alternating Hemiplegia of Childhood (AHC) zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633309
  • A rare neurological disorder4 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.
    • ]]>     2021-12-07 03:17:10 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633309     AHC Symptoms zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633408 Some early symptoms2:
    • irregular eye movements
    • difficulty swallowing
    • involuntary limb movements
    • paralysis
    Shared symptoms with other diseases4,5:
    • movement problems like Cerebral Palsy
    • learning challenges like autism
    • behavioral issues like ADHD
    • true epilepsy in at least 50% of cases
    ]]>     2021-12-07 03:17:15 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633408     Exp II Results: tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633436 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; ]]>     2021-12-07 03:17:17 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633436     Cause of AHC zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633479
  • De novo mutations4,5 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.
    • ]]>     2021-12-07 03:17:18 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633479     Urgent need for a cure zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633568
  • Diagnosis4 - onset of symptoms before reaching 18 m/o. 
  • Available drugs only alleviate the more severe symptoms3 e.g. seizures.
  • Presently, there is no cure for AHC1-5.
  • 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.
    • ]]>     2021-12-07 03:17:22 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932633568     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. zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932638929 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.
    ]]>     2021-12-07 03:20:54 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932638929     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 zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639034 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.
    ]]>     2021-12-07 03:20:59 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639034     Proposed model can work for syndromes under the umbrella of ATP1A3-Related Spectrum Disorders zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639148 ATP1A3-related ATPase dysfunction3, 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.
    ]]>     2021-12-07 03:21:03 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639148     Bibliography (used for the introduction, experiments IIIA, IIIB & IV) zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639260
  • 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.
  • https://www.tiktok.com/@jordandaniellesil?is_from_webapp=v1&is_copy_url=1&item_id=6953479054976748805
  • 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.
  • Samanta, D. (2020). Management of alternating hemiplegia of childhood: A review. Pediatric Neurology, 103, 12–20.
  • About — Cure AHC. (n.d.). Cureahc.Org. Retrieved November 12, 2021, from https://www.cureahc.org/about
    • ]]>     2021-12-07 03:21:08 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639260     Member contributions~ zshuha https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639549 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]]>     2021-12-07 03:21:21 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932639549     How AAV gene transfer works tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932648921 - 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!]]>     2021-12-07 03:27:54 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932648921     Science keeps hope alive! tahiyat01 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932654015 2021-12-07 03:31:17 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932654015 Future runzewang2 https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932682196 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.

    ]]>     2021-12-07 03:51:40 UTC https://padlet.com/tahiyat01/14oa56742y6amy1w/wish/1932682196