Ataxia

Learning Objectives

• Understand the molecular basis of frequent forms of Ataxias:

  • Explain genetic mutations underlying some SCAs and FRDA.

  • Describe the role of polyglutamine expansion (SCAs) and GAA repeat expansion (FRDA) in disease pathogenesis.

• Explore current and emerging gene therapy strategies

  • Gene Augmentation: Introduction of a functional copy of the affected gene.

  • Gene Silencing: Reducing toxic protein levels via RNAi or antisense oligonucleotides.

  • Gene Editing: CRISPR-Cas9 and other genome-editing techniques to correct mutations.

• Discuss challenges and future perspectives in gene therapy:

  • Identify key challenges such as immune responses, vector delivery efficiency, and phenotoxicity.

  • Discuss new approaches to optimize vector design, dosage control, and targeted delivery.

Ataxia

• Ataxia: “lack of order/coordination”

  • Loss of balance: with unsteady, irregular, wide-based gait.

  • Irregular and fragmented limbic movements.

  • Disrupted eye movements: not calibrated, sometime with unstable fixation, with or without double vision.

  • Slurred speech: variable speed and volume.

Autosomal Dominant and Recessive Mendelian Genes

• Autosomal dominant: Affected Parent (Aa) + Normal Parent (aa) yields offspring with genotypes Aa, Aa, aa, aa.

• Autosomal recessive: Carrier Parent (Aa) + Carrier Parent (Aa) yields offspring with genotypes AA, Aa, Aa, aa.

Spinocerebellar Ataxias (SCAs)

• Spinocerebellar Ataxias (SCAs) are progressive neurodegenerative disorders.

• Polyglutamine (PolyQ) SCAs result from CAG repeat expansions in coding regions.

• Affected individuals develop motor coordination deficits, dysarthria, and oculomotor abnormalities.

• No current cure; treatment is symptomatic.

SCA Types and Repeat Expansions

• CAG/PolyQ SCAs: SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17.

• Other Expansions: SCA8 (CTG), SCA10 (ATTCT), SCA12, SCA31 (TGGAA), SCA36 (GGCCTG).

• Non-Repeat Mutations: SCA5, SCA11, SCA13, SCA14, SCA15/SCA16, SCA20, SCA21, SCA25, SCA27, SCA28, SCA29, SCA30, SCA32, SCA34, SCA35, SCA37, SCA38, SCA39, SCA40, SCA42, SCA44, SCA45, SCA47, SCA48.

• SCAs with known chromosomal locus but unknown gene and mutation: SCA4, SCA16.

Geographical Distribution of SCAs

• SCAs have varying geographical distributions.

• Examples mentioned: SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA10, SCA14, SCA17, SCA28, SCA31, SCA36, DRPLA.

• Polyglutamine SCAs are highlighted in certain regions.

• SCAs caused by non-coding expansions or conventional mutations are also noted.

PolyQ Disorders

• PolyQ disorders are translated triplet repeat diseases.

• Gain of function: the product of the gene of interest is expressed and leads to an abnormal product.

Friedreich's Ataxia (FRDA)

• Autosomal recessive neurodegenerative disorder.

• Characterized by the progressive loss of voluntary movement coordination (ataxia) and heart enlargement.

• FRDA involves Untranslated Triplet GAA Repeat Diseases.

• Loss of function: The product of the gene of interest is not expressed or is mildly expressed.

GAA Expansion in Frataxin Gene

• GAA expansion in frataxin gene leads to loss of function.

• The mutation leads to the progressive silencing of the gene, and downstream decrease of product.

• Control state: (GAA)$_{6-36}$

• FRDA state: (GAA)$_{700-1700}$

PolyQ SCAs

• Classification: Includes SCA1, SCA2, SCA3 (Machado-Joseph Disease), SCA6, SCA7, and SCA17.

• Disease severity and onset depend on CAG repeat length.

• Common pathogenic mechanisms: protein aggregation, RNA toxicity, mitochondrial dysfunction.

Gene Therapy Strategies for PolyQ SCAs

• Gene Augmentation: Introduction of functional genes to counteract disease effects.

• Gene Silencing: RNAi, ASOs to suppress mutant gene expression.

• Gene Editing: CRISPR-Cas9 to delete or correct expanded repeats.

• Epigenetic Modulation: Restoring normal gene expression patterns.

Current Strategies for Gene Therapy Tools

• Antisense oligonucleotides (ASOs)

• Viral (AAV)-mediated gene replacement

• CRISPR Cas9 genome editing

Antisense Oligonucleotides (ASOs)

• 15-25 bases

• Single-stranded DNA fragments are combined with mRNA to form a duplex

• Preventing mRNA from being translated into protein

• Reducing the upstream signal transduction of protein expression.

Adeno-Associated Viral Vectors (AAV)

• Safe, effective, and long-term gene and protein expression.

• Organ target.

• Not known pathogenicity.

• Used for inherited disorders.

• Viral (AAV)-mediated gene replacement

CRISPR Cas9 Genome Editing

• CRISPR (clustered regulatory interspaced short palindromic repeats) systems are loci that contain multiple short direct repeats

• Can introduce sequence-specific silencing of invading foreign DNA.

Gene Therapy Definition

• Gene therapy is an experimental technique that uses genes to treat or prevent disease.

• Diseases caused by genetic defect:

  • Autosomal dominant Spinocerebellar Ataxias (SCAs)

  • Autosomal recessive ataxia (Friedreich’s Ataxia)

Gene Therapy Strategies

1. Gene Augmentation strategies or gene replacement therapy.

2. Gene Silencing Strategies.

3. Gene Edition.

Gene Augmentation in PolyQ SCAs

• Focuses on introducing genes that improve neuronal function.

• Targets neuroprotective pathways, autophagy activation.

• Examples:

  • CYP46A1 (Cholesterol Metabolism) in SCA3.

  • Beclin-1 (Autophagy Activator) in SCA3.

  • Ataxin-1-like (Competes with Mutant Ataxin-1) in SCA1.

Gene Augmentation Strategies Details

• Monogenic recessive diseases: adding one copy of the normal allele is enough for phenotype reversion.

• Monogenic dominant or complex diseases: two main mechanisms to degrade misfolded proteins: autophagy and the ubiquitin-proteosome system.

  • Activating Autophagy:

    • Re-establishment of cholesterol 24-hydroxylase (CYP46A1) showed a reduction in ataxin-3 aggregates accumulation and improved motor deficits in SCA3.

    • Lentiviral vector encoding for beclin-1 overexpressed beclin-1 in mouse models of SCA3/MJD.

    • Adeno-associated virus (AAV) vector carrying Homer-3 was used in a SCA1 model.

Gene Augmentation Strategies - Neuroprotection

• Overexpression of ataxin-1.

• Overexpression of ataxin-3

• Overexpression of endogenous calpastatin, a calpain-specific inhibitor in SCA3.

• Overexpression CRAG (collapsin response mediator protein (CRMP)- associated molecule (CRAM[CRMP-5])- associated GTPase) that facilitates the PolyQ aggregates degradation through the ubiquitinprotease pathway).

• Expression of a neuropeptide Y (NYP) (inhibitor of cell death, autophagy stimulator, anti-inflammatory effect, and increased trophic support).

Gene Augmentation Strategies Table

• Table 3 includes details on selected gene augmentation strategies studies in polyglutamine spinocerebellar ataxias.

• Includes Disease, Molecular Target, Gene Delivery System, Strategy, and References for SCA1 and SCA3.

Gene Silencing in PolyQ SCAs

• Targets disease-causing RNA to prevent mutant protein production.

• Methods:

  • RNA interference (siRNA, shRNA, miRNA)

  • Antisense oligonucleotides (ASOs)

• Examples:

  • AAV-mediated siRNA against ATXN3 (SCA3)

  • ASOs to reduce mutant Ataxin-7 in SCA7 models

Gene Silencing Strategies

• Silencing the expression of the gene containing the disease-causing mutation.

  • RNA degradation

  • Skipping the mutant exon

  • Impairing protein translation, correcting the pathological mutation, or preventing gene translation altogether.

RNA Degradation

• Endogenous RNA interference (RNAi) pathway is driven by microRNAs (miRNAs), endogenous non-coding RNAs comprised of approximately 22 nucleotides.

• miRNA and shRNA, the RNAi

MicroRNAs Table

• Includes information on Disease, microRNAs, Target, Experimental System, Delivery, and References for SCA1, SCA3/MJD, SCA6, and SCA7.

Antisense Oligonucleotides

• RNAse H-dependent: RNAse H-dependent mRNA degradation (a portion of nucleotides in the 20 position of the ASOs molecules must remain unmodified).

• RNAse H-independent: RNAse H-independent RNA modulation (completely 20-modified ASOs may be used to mediate several processes where mRNA degradation is not the outcome).

Antisense Oligonucleotides Table:

• Includes information on Disease, Target, Mechanism, Delivery Method, and References for SCA1, SCA2, SCA3/MJD, and SCA7.

Gene Editing in PolyQ SCAs

• CRISPR-Cas9 has been explored for deleting pathogenic CAG repeats.

• Study in SCA3-iPSCs demonstrated excision of expanded CAG repeats.

• Potential to permanently correct disease-causing mutations.

• Challenges include precise targeting and safety concerns.

Future Perspectives

• Ongoing advancements in gene therapy delivery and safety.

• Preclinical studies are promising, but clinical translation is needed.

• PolyQ SCAs share common mechanisms, allowing cross-disease strategies.

• Combining therapies (silencing + neuroprotection) may enhance effectiveness.

Conclusion for PolyQ SCAs

• Gene therapy offers promising approaches for PolyQ SCAs.

• Strategies include gene augmentation, silencing, and editing.

• CRISPR, ASOs, and RNAi hold significant therapeutic potential.

• Future research should focus on clinical translation and safety.

Gene Therapy Types

• Gene transplantation: Introducing a new gene into the body to help fight a disease.

• Gene correction: Inactivating, or “knocking out,” a mutated gene that is functioning improperly (to revert specific mutation in the gene of interest).

• Gene replacement: Replacing a mutated gene that causes disease with a healthy copy of the gene (to enhance the expression of the gene of interest).

Types of Gene Therapy

• Somatic gene therapy: transfer of a section of DNA to any cell of the body that doesn’t produce sperm or eggs.

  • The effects of gene therapy will not be passed on to the patient’s children.

• Germline gene therapy: the transfer of a section of DNA to cells that produce eggs or sperm.

  • The effects of gene therapy will be passed onto the patient’s children and subsequent generations.

Methods of Gene Delivery

1. Viral Vectors:

   • Adenovirus

   • Retrovirus

   • Lentivirus

   • Adeno-associated virus (AAV)

   • Herpes simplex virus (HSV)

2. Non-viral vector based:

   • Naked DNA: injection or gene gun.

   • Liposomes (cationic lipids): mix with genes

3. Ex-vivo

4. In vivo

Gene Transfer Techniques

• In vivo: Suspension containing vector is injected directly into the patient either systemically (i.v.) or directly into target tissue (e.g. malignant tumour).

• Ex vivo: Target cells (stem cells, myoblasts, fibroblasts etc) removed from the patient, treated with vector and injected back into the patient.

In Vivo Gene Therapy

• In vivo gene therapy involves introduction of therapeutic DNA directly into the patient's body.

• The DNA is introduced by cell-specific direct injection into tissue in need.

• DNA in the form of a plasmid vector is introduced by a dermal vaccination.

• Modified liposomes are not currently used for gene therapy, but they will likely be the next advancement in therapeutic gene delivery as cell-specific receptor-mediated DNA carriers.

• Once inside the body and in contact with the specifically targeted cells, the inserted DNA is incorporated into the tissue's cells where it encodes the production of the needed protein.

Ex Vivo Gene Therapy

• Ex vivo gene therapy is performed with the genetic alterations of patient's target cells happening outside of the body in a culture.

• Target cells from the patient are infected with a recombinant virus containing the desired therapeutic gene.

• These modified cells are then reintroduced into the patient's body, where they produce the needed proteins that correspond to the inserted gene.

Gene Therapy in Friedreich's Ataxia (FRDA)

GAA Expansion in Frataxin Gene (FRDA)

• Control state: (GAA)$_{6-36}$

• FRDA state: (GAA)$_{700-1700}$

Molecular Basis and Pathogenesis of FRDA

• FXN deficiency leads to mitochondrial dysfunction and iron accumulation.

• Impaired Fe-S cluster biogenesis affects cellular energy metabolism.

• Neurodegeneration primarily in spinal cord and cerebellum.

• Cardiomyopathy is a leading cause of death in FRDA patients.

Therapeutic Approaches for FRDA

• Multiple therapeutic approaches addressing FA at multiple levels (gene, cell, tissue, symptoms).

• FXN Gene mutation - GAA expansion: Gene editing, Gene silencing, Gene therapy

• Low levels of frataxin protein: Protein replacement, Stem cell therapy, Frataxin mimetics

• Mitochondrial dysfunction, oxidative stress: Mitochondrial targeting, Stress inhibitors, ROS inhibitors

• Progressive cellular degeneration: Gene-tac, Oligonucleotides, Epigenetic modifiers

• Cellular degeneration: NAD+ and exercise

• Organ and tissue dysfunction: Symptoms

Gene Therapy Strategies for FRDA

• Gene Replacement: Delivering functional FXN using viral vectors.

• Gene Silencing: RNAi or ASOs targeting FXN- silencing mechanisms.

• Gene Editing: CRISPR-Cas9 for GAA repeat correction.

• Neurotrophin Therapy: Delivery of BDNF to promote neuronal survival.

Gene Therapy Delivery Methods for FRDA

• Viral Vectors: AAV9 and AAVrh10 efficiently transduce CNS and heart.

• Administration Routes:

  • Intravenous (systemic): Used in Zolgensma for SMA.

  • Intrathecal (IT): Directly reaches spinal cord, used in ALS trials.

  • Intracerebroventricular (ICV): Best CNS-wide distribution.

  • Intraparenchymal: Precise cerebellar targeting but invasive.

Challenges and Future Perspectives in FRDA Gene Therapy

• Immune Response: AAV vectors can trigger immune reactions.

• Phenotoxicity: Excessive FXN or BDNF overexpression can be toxic.

• Gene Expression Regulation: Need for controlled FXN expression to avoid toxicity.

• Clinical Trials: Need for robust safety and efficacy data before clinical translation.

Conclusion for FRDA Gene Therapy

• Gene therapy is a promising approach for Friedreich’s Ataxia.

• AAV-mediated FXN delivery has shown preclinical success in FA models.

• New gene editing and neurotrophin strategies offer additional therapeutic avenues.

• Ongoing research is focused on optimizing safety, delivery, and clinical translation.

AAVS in-vivo approach for neurodegenerative diseases.

• Routes of Administration:

  • Intracerebroventricular (ICV)

  • Intrathecal (IT)

  • Systemic Intravenous (IV)

  • Intra-cisterna magna (ICM)

  • Deep cerebellar nuclei Injection

  • Cerebellar cortex injection

  • 4th Ventricle direct injection

FRDA Gene Therapy Treatments - Table

• Table includes various treatments, doses, animal models, age, delivery route, major improvements and references.

• HSV1-hFXN

• AAVrh10-CAG-hFXN-HA

• HSV-BDNF

• AAV9-CAG-hFXN-HA

• AAV9-hFXN

• AAVrh.10hFXN

Additional Notes on FRDA Gene Therapy

• Multiple approaches have been studied (e.g., antioxidants, epigenetic drugs, etc) for the treatment of FRDA, and gene therapy is one of the most promising.

• A point of major concern in gene therapy is phenotoxicity or the toxicity associated with the overexpression of the transgene.

• Studies in cultured human cells have demonstrated that FXN overexpression also leads to oxidative stress and toxicity, in a similar way to frataxin deficiency, underscoring the importance of physiological levels of FXN to confer a therapeutic effect.

Challenges in Gene Therapy

• Short-lived nature of gene therapy

• Difficult to treat multi gene or multi factorial disease

• Inserting gene into correct cells.

• Controlling gene expression. Possibility of over expression

• Damage to the host gene

• Acquirement of virulence

• Chance of inducing a tumour (insertional mutagenesis)