Gene Therapy for Neuromuscular Diseases

Slide 1 · Gene Therapy for Neuromuscular Diseases

Pathogenic mechanism • molecular intervention • clinicopathologic interpretation

From molecular lesion to therapeutic design, tissue response, and clinical evidence

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning outcomes

Match loss-of-function and toxic gain-of-function mechanisms to appropriate genetic treatment classes.

Explain AAV cargo limitations, mini-dystrophin, immune barriers, and hepatic toxicity.

Interpret exon skipping, read-through, RNA silencing, and genome editing without equating molecular response with proven benefit.

Evaluate genotype eligibility, tissue biology, clinical endpoints, and contemporary safety changes.

Slide 3 · Therapeutic logic begins with pathogenic mechanism

Molecular pathogenic lesion

Loss of functional protein

Gene replacement / gene modulation

Toxic gene product or RNA

Targeted knockdown / silencing

Disease-causing DNA sequence

Genome correction: Investigational

Slide 4 · Four therapeutic verbs mean different things

Approach

What changes

What remains

Replace

Introduce a functional transgene

Native mutant locus usually remains

Modulate

Change RNA splicing or translation

Underlying DNA variant persists

Silence

Reduce pathogenic RNA/protein

Mutated DNA generally persists

Correct

Edit the causative DNA sequence

Potentially alters endogenous locus

Slide 5 · DNA → RNA → protein: Three sites of action

DNA

Variant

Gene addition / editing

RNA

Transcript

Exon skipping / silencing

Protein

Product

Read-through / restored protein

Tissue

Phenotype

Functional outcome needs testing

Slide 6 · Genotype-specific treatment is not optional detail

A mutation class may predict whether gene addition, splice modulation, or read-through is mechanistically plausible.

Exon skipping is defined by the exact DMD exon configuration; A diagnosis of Duchenne muscular dystrophy alone is insufficient.

Toxic gain-of-function is distinct from simple protein deficiency.

Molecular confirmation precedes claims of patient eligibility.

Slide 7 · Broad replacement versus narrow mutation matching

Gene replacement

Sequence-specific modulation

Designed to supply functional protein independently of some variant locations.

Still limited by vector capacity, tissue delivery, and immune biology.

Requires a variant that is amenable to the particular RNA intervention.

Can preserve endogenous gene regulation but does not repair DNA.

Slide 8 · Dystrophinopathy: A model for genetic precision

Duchenne and Becker muscular dystrophies result from loss-of-function DMD variants on the X chromosome.

Dystrophin is a structural component of the sarcolemmal protein complex.

The amount and functionality of remaining dystrophin help explain phenotype severity.

This genotype–protein–phenotype relationship motivates targeted treatment.

Slide 9 · Dystrophin links muscle cytoskeleton to extracellular matrix

Extracellular matrix / laminin

Sarcolemmal dystroglycan–sarcoglycan complex

Intracellular cytoskeleton

Dystrophin provides linkage and stability

Slide 10 · Loss of dystrophin destabilizes the sarcolemma

Defective

Dystrophin

Loss of mechanical linkage

Membrane

Vulnerability

Contraction-related membrane injury

Calcium

Influx

Damage-triggering intracellular events

Myofiber

Necrosis

Progressive tissue injury

Slide 11 · Disrupted reading frame often produces severe DMD

Frameshift variants disrupt the coding frame and commonly abolish useful dystrophin synthesis.

The full-length DMD gene encodes a very large protein.

Severe Duchenne disease is associated with absent dystrophin rather than merely reduced normal function.

Frameshift status explains why RNA-level restoration is a rational target.

Slide 12 · Becker dystrophy demonstrates partial protein function

Duchenne pattern

Becker pattern

Absent or profoundly deficient dystrophin

Earlier, more severe progressive muscle damage

Truncated dystrophin with retained activity

Often later onset and less severe progression

Slide 13 · Restoring some function is biologically meaningful

No useful

Dystrophin

Typical severe loss-of-function

Shortened

Functional form

Exon skipping may yield this

Improved membrane

Support

Functional effect is not binary

Potentially milder

Phenotype

Clinical size of benefit must be proven

Slide 14 · Young DMD muscle: Necrosis and regeneration coexist

Muscle damage outpaces compensatory repair.

Biopsies may show segmental myofiber necrosis and clusters of regenerating fibers.

Myofiber size varies even while fascicular architecture remains recognizable.

Myophagocytosis may accompany degeneration without prominent primary inflammation.

Slide 15 · Advanced DMD muscle: Tissue replacement becomes dominant

Repeated

Necrosis

Persistent injury

Repair

Capacity exceeded

Incomplete regeneration

Endomysial

Fibrosis

Architectural distortion

Fatty tissue

Replacement

Reduced contractile muscle mass

Slide 16 · Dystrophin immunostaining has a distinct interpretive role

Finding

Pathology interpretation

Therapeutic caution

Absent sarcolemmal stain

Supports severe dystrophin deficiency

Baseline expression is not a treatment endpoint

Reduced/patchy expression

May reflect residual protein

Expression pattern requires careful validation

Post-treatment expression

Evidence of protein production

Does not independently prove functional benefit

Slide 17 · Muscle biopsy and sequencing answer different questions

Molecular analysis

Tissue analysis

Identifies pathogenic variant and reading-frame consequences.

Determines potential exon-skipping or nonsense-mutation eligibility.

Documents degeneration, fibrosis, and dystrophin expression.

Provides context for protein-expression biomarkers.

Slide 18 · Functional decline involves more than limb strength

DMD begins with proximal weakness but progresses to major mobility limitation.

Respiratory muscles may become weak, producing impaired ventilation.

Cardiac dystrophin deficiency contributes to cardiomyopathy and arrhythmias.

A tissue-specific benefit should not be assumed to protect every affected organ.

Slide 19 · Gene delivery addresses one step in a longer chain

Vector reaches

Target tissue

Biodistribution

Transgene is

Expressed

Molecular expression

Protein restores

Needed function

Tissue-level function

Clinical course

Changes

Patient-important outcome

Slide 20 · AAV is a vehicle for DNA delivery

Adeno-associated virus vectors can carry DNA constructs encoding a functional protein.

The delivered construct is not automatically a correction of the patient’s genomic locus.

Therapeutic success depends on both cargo design and target-tissue expression.

AAV delivery raises vector-specific immune and safety constraints.

Slide 21 · AAV delivery: Vector → target cell → gene expression

Engineered

AAV vector

Package transgene

Entry into

Target cell

Tissue exposure

Delivered DNA

Is expressed

Transcription / translation

Therapeutic

Protein produced

Downstream activity must be assessed

Slide 22 · AAV capacity conflicts with the size of DMD

AAV carrying capacity

Approximate AAV packaging limit

4.7 kb

DMD coding sequence

14 kb

The 14 kb coding sequence exceeds a single conventional AAV cargo budget.

Slide 23 · A size mismatch forces construct redesign

The textbook contrasts approximately 4.7 kb AAV packaging capacity with roughly 14 kb of DMD coding sequence.

Full-length coding sequence cannot fit in a conventional single AAV cassette.

A smaller dystrophin-derived transgene offers a practical delivery concept.

The engineered protein is not the intact native dystrophin molecule.

Slide 24 · Mini-dystrophin: Selective retention of useful function

Full-length dystrophin

Engineered abbreviated construct

The native protein has a coding sequence too large for single conventional AAV packaging.

Multiple structural interactions contribute to muscle stability.

Removes sequence to accommodate vector constraints.

Must retain enough relevant functionality for a therapeutic effect.

Slide 25 · Replacement does not recreate the original gene

Question

Gene addition using AAV

Genome editing concept

DNA locus

Mutant native locus remains

Causative sequence is the target

Therapeutic expression

Introduced DNA encodes a protein

Edited locus may produce corrected product

Major hurdle

Cargo/delivery/immune effects

Editing accuracy and delivery

Status

Clinically implemented for DMD

Further from clinical implementation

Slide 26 · Protein produced ≠ normal tissue restored

Mini-dystrophin

Expression

Molecular endpoint

Membrane support

May improve

Proposed mechanism

Established fibrosis

May persist

Pre-existing irreversible pathology

Clinical course

Remains uncertain

Requires long-term observation

Slide 27 · Neutralizing antibodies can impede AAV

Naturally occurring antibodies against AAV may neutralize the viral vector.

An otherwise suitable transgene cannot act if delivery to its target is blocked.

Serologic vector considerations differ from the causative disease variant.

Eligibility therefore has both disease-genetic and delivery-related dimensions.

Slide 28 · Immune responses may target the vector itself

AAV capsid

Exposure

Viral proteins

Host immune

Recognition

Immune barriers

Inflammatory

Response

Potential adverse reactions

Safety / expression

Consequences

Monitor beyond the intended transgene

Slide 29 · Immune responses may target the new protein

The transgene can produce a protein that the host has not previously expressed in normal amounts.

The expressed transgene product itself may become an immune target.

Immunity can complicate safety as well as durability of expression.

Gene construct design and host biology cannot be evaluated independently.

Slide 30 · Hepatotoxicity is a major AAV limitation

Mechanistic rationale

Clinical interpretation

Systemic vector administration exposes organs beyond skeletal muscle.

Hepatotoxicity as a common AAV-related adverse effect.

Liver safety requires a separate risk assessment from muscle efficacy.

Severe post-treatment liver injury can outweigh a plausible molecular benefit.

Slide 31 · AAV platform: Delivery advantages and constraints

Potential advantage

Mechanistic constraint

Introduces new protein-coding DNA

Limited payload capacity

Can reach biologically relevant cells

Pre-existing neutralizing antibodies

Targets a causal protein deficit

Capsid / transgene immune responses

Can create measurable protein expression

Hepatic toxicity and uncertain long-term benefit

Slide 32 · AAV evidence must be interpreted in layers

After therapy: What was demonstrated?

Construct detected / delivered

Distribution or exposure

Protein measured in tissue

Molecular target engagement

Durable patient function improved

Clinically meaningful benefit

Slide 33 · Protein expression is a surrogate endpoint

Demonstrating newly produced dystrophin supports biological activity of the construct.

The extent, distribution, and functionality of expressed protein all matter.

Improved protein expression does not automatically establish improved walking or cardiopulmonary outcomes.

Long-term functional benefit must be tested independently.

Slide 34 · Long-term AAV effectiveness remains an empirical question

The long-term benefits of DMD mini-dystrophin delivery remain to be determined.

Short-term biochemical effects cannot establish lifelong muscle protection.

Progressive baseline disease and already established tissue injury complicate follow-up.

Clinical trials and extended surveillance address questions that mechanistic plausibility cannot settle.

Slide 35 · Exon skipping intervenes during RNA processing

Mutated DMD

Pre-mRNA

Reading frame disturbed

Sequence-matched

Antisense oligo

Binding at RNA level

Selected exon

Is skipped

Alternative splicing

Frame-restored

MRNA translated

Shortened dystrophin may be produced

Slide 36 · Frameshift and reading-frame rescue

Illustrative pre-mRNA before splice modification

EXON A

EXON B (targeted)

EXON C

ASO-mediated splice redirection

EXON A

EXON B excluded

EXON C

Schematic: Exon exclusion can restore a frame only for a variant with suitable exon lengths.

Slide 37 · Exon skipping does not repair the DNA variant

Changes

Does not change

Splicing pattern of the DMD transcript

Potential production of shortened partly functional dystrophin

The pathogenic variant in genomic DNA

The need for variant-specific therapeutic selection

Slide 38 · Specific exons identify eligible molecular subgroups

Exon 45-, 51-, and 53-skipping approaches for selected DMD variants.

The relevant criterion is whether the individual variant is amenable to the specified exon skip.

The exon number identifies the target of the oligonucleotide—not every mutation located within that exon.

Variant annotation and reading-frame prediction are therefore essential.

Slide 39 · Exon skipping is only relevant to amenable variants

Confirmed pathogenic DMD variant

Frame disrupted; Exon skip can restore frame

Evaluate exact matching antisense approach

Variant not amenable to targeted exon skip

This exon-skipping strategy is not suitable

Variant is a premature stop codon

Read-through is a different proposed mechanism

Slide 40 · Why exon skipping evokes a Becker-like mechanism

Out-of-frame

Transcript

Usually no useful full protein

Exon removed

At RNA level

RNA splice modulation

In-frame shortened

Transcript

Frame compatibility restored

Partly functional

Dystrophin

Analogous in principle to milder variants

Slide 41 · RNA modulation and DNA delivery have distinct liabilities

Feature

AAV gene addition

Antisense exon skipping

Intervention site

Introduced DNA

Endogenous pre-mRNA

Main selection issue

Vector/construct and host factors

Exact amenable DMD genotype

Product

Engineered transgene protein

Splice-altered endogenous dystrophin

Important caveat

Immune/hepatic toxicity

Restoring frame need not guarantee clinical effect

Slide 42 · Skipping an exon is not silencing the gene

Exon skipping

Gene knockdown

Redirects splice-site use in a particular transcript.

Aims to permit production of a useful shortened protein.

Reduces abundance of pathogenic RNA or protein.

Aims to diminish toxic gain-of-function.

Slide 43 · Nonsense variants disrupt translation

Normal coding

Sequence

Open reading frame

Premature stop

Codon

Nonsense mutation

Translation

Terminates

Truncated translation

Full protein

Not produced

Loss of function follows

Slide 44 · Read-through aims to overcome premature stop signals

Nonsense mutation

In mRNA

Distinct subgroup

Read-through

Pharmacologic effect

Ribosomal mechanism

Translation may

Continue

Not DNA repair

More full-length

Product possible

Clinical effectiveness requires evidence

Slide 45 · Nonsense read-through is subgroup-specific

That nonsense variants occur in approximately 10–15% of Duchenne cases.

This mutation class is biologically different from an out-of-frame deletion.

Read-through drugs aim to modify protein synthesis, not exon selection.

Patient-specific biological eligibility does not establish clinical efficacy or authorization.

Slide 46 · A mechanistic rationale is not regulatory proof

Plausible mechanism

Evidence required

A ribosome could translate past a premature termination signal.

Restored product is a testable molecular hypothesis.

Clinical benefit must be reproducible in affected patients.

Authorization depends on regulatory review and can change over time.

Slide 47 · Toxic gain-of-function demands a different objective

Disease caused by toxic expression

Reduce toxic RNA

Antisense oligonucleotide silencing

Reduce toxic protein production

RNA interference or other gene knockdown

Replace a missing protein

May fail to address the toxic mechanism

Slide 48 · Dominant disease may retain a normal allele

Dominant inheritance may arise from toxic gain-of-function rather than simple haploinsufficiency.

Supplying an extra wild-type copy does not necessarily eliminate the toxic product.

A reduction strategy aims to diminish pathogenic expression.

Whether allele-selective versus broader suppression is desirable depends on disease biology.

Slide 49 · RNA interference concept: Reduce pathogenic transcript

Pathogenic

RNA transcript

Toxic gain-of-function

RNA-directed

Silencing

Target-complementary process

Reduced target

RNA abundance

Knockdown

Reduced toxic

Product

Potential tissue benefit

Slide 50 · Antisense oligonucleotides can serve two roles

Therapeutic use

RNA effect

Intended result

Exon skipping

Redirect pre-mRNA splicing

Produce partly functional shorter protein

Transcript silencing

Decrease pathogenic RNA

Reduce toxic gain-of-function

“Antisense” describes a molecular tool class, not one uniform biological outcome.

Slide 51 · Myotonic dystrophy illustrates toxic RNA biology

Expanded CTG

Repeat in DMPK

DM1 genetic lesion

Repeat-containing

RNA accumulates

Toxic RNA behavior

Splicing regulators

Sequestered

Loss of normal splicing control

Alternative splicing

Is altered

Multisystem manifestations

Slide 52 · DM1 provides a rationale for RNA-directed therapy

Underlying lesion

Logical intervention target

Expanded repeat-containing transcript binds splicing regulators.

CLCN1 alternative splicing contributes to myotonia.

Lower the toxic transcript or interrupt its pathological interactions.

This is a mechanistic rationale—not an approved therapy claim.

Slide 53 · Silencing and exon skipping must not be conflated

Question

Exon skipping

Knockdown

Main problem

Loss of usable protein

Toxic product

Desired RNA change

Different splice isoform

Lower target RNA abundance

Expected protein effect

Partially restore activity

Decrease toxic protein or RNA

Typical mechanism in source

Selected DMD variants

Dominant toxic gain-of-function

Slide 54 · CRISPR/Cas9 represents a gene correction strategy

Identify DNA

Lesion

Mutation-specific target

Introduce editing

Machinery

Gene-editing platform

Modify pathogenic

Sequence

Correction is intended

Assess genomic and

Functional effects

Research-stage in source

Slide 55 · Genome editing and gene addition differ fundamentally

Question

Gene addition

Gene correction

Molecular substrate

DNA introduced by a vector

Endogenous DNA sequence

Typical intention

Compensate for loss of function

Alter causal sequence

Primary limitation

Vector/cargo/immune barriers

Farther from clinical implementation

Shared requirement

Demonstrate safety and patient benefit

Demonstrate safety and patient benefit

Slide 56 · Genome editing needs target and consequence verification

A DNA change must be characterized at the intended target locus.

An altered sequence matters only if it restores a biologically relevant function.

Delivery to the affected tissues remains essential.

Genome correction with CRISPR/Cas9 farther from routine clinical implementation.

Slide 57 · Genetic eligibility and clinical eligibility are different

Genetic matching

Clinical matching

Causative gene and pathogenic variant confirmed.

Predicted relationship between variant class and treatment mechanism.

Disease stage and affected organ systems characterized.

Product-specific indication and safety restrictions independently checked.

Slide 58 · Pathology gives context to therapeutic biomarkers

Early muscle disease can retain fascicular architecture despite substantial necrosis and regeneration.

Advanced fibrosis and fatty replacement represent loss of functioning muscle tissue.

Dystrophin immunostaining indicates protein expression but not its entire functional effect.

Post-treatment tissue findings should be interpreted against disease stage.

Slide 59 · Muscle, heart, and respiratory outcomes are distinct

Patient-important treatment outcome

Skeletal muscle

Strength, ambulation, daily function

Respiratory system

Ventilatory muscle performance

Heart

Cardiomyopathy and rhythm outcomes

Slide 60 · Clinical endpoints must test the causal hypothesis

Molecular

Target engagement

Gene/protein assay

Tissue-level

Response

Morphology / biochemical effect

Functional

Performance

Patient-important outcomes

Durability and

Safety

Longitudinal evidence

Slide 61 · AAV DMD therapy: Regulatory context changed in 2025

Robbins baseline statement

FDA update — Nov 2025

An AAV abbreviated-dystrophin strategy had gained approval.

The general term mini-dystrophin.

Elevidys specifically encodes engineered micro-dystrophin.

FDA indication: Ambulatory DMD, ≥4 years, with confirmed DMD mutation.

Slide 62 · Fatal liver injury reshaped the Elevidys indication

FDA reported fatal acute liver failure in non-ambulatory DMD patients treated with Elevidys.

The November 2025 FDA labeling update removed the non-ambulatory indication.

The FDA boxed warning highlights serious liver injury and acute liver failure, including fatal outcomes.

Current use must follow the latest product-specific prescribing information.

Slide 63 · Exon-skipping regulatory status is not proof of benefit

An FDA July 2026 advisory briefing identified four approved DMD antisense drugs targeting exons 45, 51, or 53.

The document reports that these approvals remain under the accelerated-approval pathway.

The briefing states that clinical benefit had not yet been verified for these four products.

Molecular dystrophin production should therefore be separated from demonstrated patient benefit.

Slide 64 · EU nonsense read-through status changed in 2025

Textbook historical claim

EMA/EC current correction

Ribosomal read-through medication achieved European approval for nonsense DMD.

The statement describes a former authorization context.

The European Commission did not renew Translarna (ataluren) authorization on 28 March 2025.

The EMA stated that clinical effectiveness had not been confirmed.

Slide 65 · Spinal muscular atrophy: Another loss-of-function model

Spinal muscular atrophy (SMA) involves pathogenic SMN1 variants and loss of motor neurons.

The therapeutic target differs from dystrophin deficiency in the muscle fiber.

FDA-approved AAV gene therapies now provide a contemporary example of gene addition in motor-neuron disease.

SMA therefore illustrates gene addition directed at motor-neuron disease rather than primary myofiber disease.

Slide 66 · FDA-approved SMA gene therapy populations differ

Therapy

FDA population

Principle

Zolgensma (onasemnogene abeparvovec-xioi)

Pediatric SMA, <2 years, biallelic SMN1 mutations

AAV-delivered gene therapy

Itvisma (onasemnogene abeparvovec-brve)

SMA, age ≥2 years, confirmed SMN1 mutation

AAV-delivered gene therapy

Product-specific FDA indications; Consult the latest prescribing information.

Slide 67 · Case exercise: Identify a strategy for an out-of-frame DMD variant

Teaching vignette (hypothetical)

An affected child has confirmed DMD frameshift disruption with absent dystrophin on muscle immunostaining.

What pathogenic mechanism is operating?

What extra variant information is needed before selecting exon skipping?

Why would a full-length DMD gene not fit in conventional single AAV?

Loss of function → assess exact exon-skipping amenability; AAV limit ≈4.7 kb versus ≈14 kb DMD coding sequence.

Slide 68 · Case exercise: Choose a strategy for toxic repeat RNA

Teaching vignette (hypothetical)

A multisystem myotonic disorder is associated with an expanded repeat-containing transcript that sequesters splicing regulators.

Is simple gene replacement likely to remove the toxic transcript?

Which RNA-directed strategy follows from the mechanism?

What finding would support target engagement?

Knockdown/silencing is the mechanistic candidate; Assess reduced toxic RNA and downstream splicing correction. Do not infer regulatory approval.

Slide 69 · Mechanism, evidence, and safety are separate decisions

Should this approach be used?

Biological fit

Correct lesion, tissue, intervention

Evidence strength

Patient benefit beyond surrogate markers

Net clinical suitability

Safety, indication, disease stage

Slide 70 · Three conclusions to retain

Select genetic treatments from the molecular pathogenesis: Replace, modulate, silence, or correct.

AAV-mediated DMD treatment is constrained by cargo size, immunity, hepatotoxicity, and uncertain long-term effect.

Exon skipping, read-through, and knockdown differ in target and mutation specificity.

Clinical efficacy and authorization status require evidence beyond mechanistic plausibility; Relevant regulatory statements have changed.

Slide 71 · Source references and verification boundary

Robbins, Cotran & Kumar. Pathologic Basis of Disease, 11th ed. (2026), ch. 27, pp. 1121–1125.

FDA: Elevidys safety/label revision (14 Nov 2025); DMD antisense briefing (29 Jul 2026).

EMA: Translarna non-renewal (28 Mar 2025).

FDA: SMA gene therapy product pages, including Itvisma (24 Nov 2025).

Regulatory status is time-sensitive; reassess current product labels and agency decisions.