consistent-delivery-adeno-associated-virus-via-lateral-tail-vein

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...

National Institutes of Health

University College London

Single nucleotide variants that alter splice sites or splicing regulatory elements can lead to the skipping of exons, retention of introns, or insertion of pseudo-exons (PE) into the mature mRNA transcripts. When translated, these changes can disrupt the function of the synthesized protein. Splice-switching antisense oligonucleotides (ASOs) are synthetic, modified nucleic acids that can correct these aberrant splicing events. They are currently in active clinical development for a number of conditions and have been approved by regulatory agencies for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy and spinal muscular atrophy. We have previously reported that splice-switching ASOs effectively skip a pathogenic PE that causes Ullrich congenital muscular dystrophy (UCMD). This erroneous PE insertion is caused by a deep-intronic variant located within intron 11 of COL6A1 (c.930+189 C>T). Here, we describe the detailed protocols and workflow that our labs have used to assess the efficacy of ASOs to skip this PE in vitro. The protocols include designing ASOs; isolating, culturing, and transfecting fibroblasts; extracting RNA and protein; and validating splicing correction at the mRNA and protein levels using quantitative reverse transcription PCR (qRT-PCR) and western blot assays, respectively.

Exon-Skipping for a Pathogenic COL6A1 Variant in Ullrich Congenital Muscular Dystrophy.

Collagen VI-related dystrophies (COL6-RDs) manifest with a spectrum of clinical phenotypes, ranging from Ullrich congenital muscular dystrophy (UCMD), presenting with prominent congenital symptoms and characterised by progressive muscle weakness, joint contractures and respiratory insufficiency, to Bethlem muscular dystrophy, with milder symptoms typically recognised later and at times resembling a limb girdle muscular dystrophy, and intermediate phenotypes falling between UCMD and Bethlem muscular dystrophy. Despite clinical and immunohistochemical features highly suggestive of COL6-RD, some patients had remained without an identified causative variant in ,  or . With combined muscle RNA-sequencing and whole-genome sequencing we uncovered a recurrent,  deep intronic variant in intron 11 of  (c.930+189C>T) that leads to a dominantly acting in-frame pseudoexon insertion. We subsequently identified and have characterised an international cohort of forty-four patients with this  intron 11 causative variant, one of the most common recurrent causative variants in the collagen VI genes. Patients manifest a consistently severe phenotype characterised by a paucity of early symptoms followed by an accelerated progression to a severe form of UCMD, except for one patient with somatic mosaicism for this  intron 11 variant who manifests a milder phenotype consistent with Bethlem muscular dystrophy. Characterisation of this individual provides a robust validation for the development of our pseudoexon skipping therapy. We have previously shown that splice-modulating antisense oligomers applied  effectively decreased the abundance of the mutant pseudoexon-containing COL6A1 transcripts to levels comparable to the  scenario of the somatic mosaicism shown here, indicating that this therapeutic approach carries significant translational promise for ameliorating the severe form of UCMD caused by this common recurrent  causative variant to a Bethlem muscular dystrophy phenotype.

The recurrent deep intronic pseudoexon-inducing variant  c.930+189C>T results in a consistently severe phenotype of COL6-related dystrophy: Towards clinical trial readiness for splice-modulating therapy.

Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating,  variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics . The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous   mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While  mice do not show any phenotype compared to wildtype animals,   and   mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the  N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other  pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

Fady Guirguis

Neuromuscular and Neurogenetic Disorders of Childhood Section,
National Institute of Neurological Disorders and Stroke,
Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke

National Institutes of Health

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice