Lecture 14 - Potential Therapies for NMD

Most clinical treatments

Steroids, physiotherapy, orthopaedic input, cardiac surveillance, ventilatory support

Challenges of designing a theory

Muscle, injection will only transduce cells only within a couple centimetres of injection, multiple injections needed, treatment would need to begin very early

Ideal therapy

Long-term delivery of missing gene or persistent gene correction

Strategies for rational therapy design

Knowledge of disease causing gene mutation, understand of pathway, pathobiology, disease mechanism, appropriate model for screening/testing putative therapies, knowledge of different therapeutic strategies

Potential therapeutic strategies

Gene replacement therapy, antisense oligonucleotides, up regulation therapy, precise correction or deletion of mutation, RNA interference, read through stop codons

Gene replacement therapy viruses

Adenovirus, adeno-associated virus, retrovirus and lentivirus

Adenoviral vectors

Popular due to large coding capacity, although caused an unexpected death A

Adeno-associated virus

Vector of choice, not associated with human disease, cannot replicate itself, can only accommodate 4.7kb, effectively transducer both skeletal and cardiac muscle, vascular delivery possible with certain serotypes G

Gene replacement therapy pros

Can persist as episomes for a long time in nucleus or can integrate into genome

Gene replacement therapy cons

Immune response can be mounted, difficult to produce enough quantities, can’t incorporate large genes

Therapy limitations

Normal protein expressed from one WT allele, defective protein from mutant allele

Exon skipping

Most applicable to mutations which introduce a premature stop codon in repetitive regions of large genes

Correction with CRISPR/Cas9

Immunogenicity of bacteria not tested long-term, NHEJ much more frequency than HDR, ethical considerations resounding editing germline cells, off target effects difficult to screen for a potentially catastrophic

Allele-specific deletion of dominant genes

Uses SNPs which add NGG PAM site to mutant allele/chromosome only; will only cut out mutant allele and keep normal allele

Upregulation

Enhance expression of a similar gene which could perform the same function, for recessive diseases

Loss of function

Gene replacement, ASO exon skipping, up regulation

Gain of function

Erase and replace, ASO knockdown, allele-specific deletion

Dominant or recessive

Correction