AAVantgarde has two proprietary, AAV-based large gene delivery platforms.

Our proprietary technology is focused on both leveraging DNA recombination, named dual hybrid; and leveraging protein trans-splicing, named AAV intein. The company is validating the platforms in two lead programs: Usher syndrome type 1B associated retinitis pigmentosa (USH1B), using dual hybrid; and Stargardt disease, using AAV intein.

AAV gene therapy has been limited by transgene capacity, and the Company’s platforms enable delivery of large genes to tissue and cells in vivo; something that could potentially extend into many disease areas.

AAV Dual Hybrid Platform

This platform uses two AAV vectors, each containing one half of an expression cassette encoding for large therapeutic transgene and works at the cell nucleus level, recombining the two halves of the transgene back into a single one within the cell.

In the AAV dual hybrid strategy, a splice donor (SD) signal is placed at the 3′ end of the 5′-half vector encoding for the 5’half of the therapeutic transgene and a splice acceptor (SA) signal is placed at the 5′ end of the 3′-half vector encoding for the 5’half of the therapeutic transgene. Moreover, in the dual hybrid, an highly recombinogenic (HR) region (proprietary of AAVantgarde) is placed downstream and upstream to the SD and SA signal respectively, with the aim to increase the recombination between the two AAV genomes. Upon co-infection of the same cell by the dual AAV vectors, the inverted terminal repeat (ITR) and HR-mediated head-to-tail concatemerization of the two halves leads to the formation of a single AAV genome that is then transcribed into RNA followed by splicing which removes the unwanted recombinogenic sequences (ITR and HR), and leads to the production of a mature full-length mRNA that is then translated in a full-size functional protein.

This technology translates into an efficient recombination that generates therapeutically meaningful protein levels in animal models and it is a versatile approach.

Ref: Trapani I., Colella P., et. al. Effective delivery of large genes to the retina by dual AAV vectors. Embo Molecular Medicine, February 2014.

AAV-Intein platform

This platform uses two AAV vectors, each containing an independent expression cassette encoding one of the 2 halves of a target protein flanked by short sequences, named as split inteins. It works by protein trans-splicing within the cell and has demonstrated a very efficient recombination to deliver therapeutically meaningful protein levels.

Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute large proteins. Within this platform, we have demonstrated that delivery of multiple AAV vectors, each encoding one of the fragments of target proteins flanked by short split inteins, results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein–mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

Ref: Tornabene P., Trapani I., et al. Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Science Translational Medicine, May 2019.