Split intein-mediated protein trans-splicing to express large dystrophins

Area of Science:

• Molecular Biology
• Gene Therapy
• Biochemistry

Background:

• Adeno-associated virus (AAV) vectors are promising for gene replacement therapy.
• AAV's limited packaging capacity (approx. 4.7 kb) restricts its use for large genes, like dystrophin (14 kb mRNA) in Duchenne muscular dystrophy.
• Current micro-dystrophin strategies face limitations.

Purpose of the Study:

• To develop a novel method for expressing large dystrophins using split inteins to overcome AAV packaging limitations.
• To evaluate the efficacy of expressing large dystrophins via split intein-mediated protein trans-splicing in dystrophic mouse models.
• To compare the functional outcomes of large dystrophins versus micro-dystrophins in vivo.

Main Methods:

• Utilized split inteins to mediate protein trans-splicing, enabling the assembly of large dystrophins from two or three fragments.
• Delivered multiple AAV vectors encoding these fragments into dystrophic mice.
• Employed the myotropic AAVMYO vector for efficient, body-wide striated muscle transduction at low doses.

Main Results:

• Successfully generated large midi-dystrophins and full-length dystrophins through split intein-mediated trans-splicing.
• Demonstrated robust expression of large dystrophins and significant physiological improvements in dystrophic mice treated with 2-3 AAVs.
• Achieved body-wide striated muscle expression of large dystrophins with substantial physiological correction in mice using low doses of AAVMYO.

Conclusions:

• The split intein approach effectively overcomes AAV packaging constraints for expressing large therapeutic proteins like dystrophin.
• Large dystrophins show clear functional superiority over micro-dystrophins, offering a more promising therapeutic strategy.
• This method holds potential for treating Duchenne and Becker muscular dystrophy and other genetic disorders involving large genes exceeding AAV capacity.