Spatial biology gives a clear view on gene therapy

Gene therapy is becoming a reality for the treatment of several conditions, but preclinical evaluation still presents a challenge¹.

Adverum Biotechnologies overcame these hurdles to achieve FDA orphan drug approval for ADVM-062, an AAV-based gene therapy candidate for the treatment of blue cone monochromacy (BCM), an inherited eye disease.

BCM is caused by an absence of L- and M- opsins in the fovea of the eye. It’s an ideal candidate for gene therapy, but a lack of appropriate disease models presented a problem.

“Non-human primates share almost identical L- and M- opsin gene sequences,” explains Julio Nieves, Adverum’s associate director of imaging. “This makes it impossible, using conventional immunohistochemistry, to detect vector activity or local production of transgenic human opsin against background noise.”

The team turned to BaseScope, one of the portfolio of products developed by Advanced Cell Diagnostics (ACD), a brand of Bio-Techne, using its spatial in situ hybridization technology platform, RNAscope.

ACD offers two suites of products for different applications. The first uses RNAscope and BaseScope in situ hybridization and is designed for AAV-based gene therapy, allowing single-molecule detection of both the episomal vector DNA, and transgene mRNA expression. The second is RNAscope Plus smRNA-RNA assays that is aimed at researchers developing oligonucleotide therapies using small RNA, antisense oligonucleotides (ASOs) or RNA interference (RNAi).

Selective amplification of single RNA molecules

RNAscope uses a proprietary probe design strategy where two independent ZZ probes simultaneously hybridize to a target sequence to amplify a chromogenic or fluorescent signal². This ensures selective amplification of on-target signals, while reducing non-specific signals. The method detects individual RNA molecules with high specificity in single cells, enabling precise confirmation of cell tropism and quantification of vector delivery and transgene expression.

“Our goal is to empower researchers with innovative, multiomic spatial biology solutions to accelerate the development of next-generation therapeutics and improve lives,” says Michaeline Bunting, senior director of product management for spatial biology at Bio-Techne. “We can provide highly specific probes to any gene therapy vector or RNA/oligonucleotide therapeutic, enabling researchers to accurately visualize and characterize tissue biodistribution and therapeutic efficacy in any preclinical model system.”

While Adverum used BaseScope to evaluate its AAV-based gene therapy, RNAscope Plus smRNA-RNA assays combine two chemistries that allow for the simultaneous detection of a therapeutic ASO or siRNAs and multiple target mRNAs in the same samples. Researchers can visualize biodistribution and track changes in the expression of genes they want to modulate in target cells.

Optimizing safety and expression

Adverum needed the full length wild-type human opsin transgene nucleotide sequence to ensure optimal expression and safety, but detection was challenging. “We opted for BaseScope because of the size of the regions we’re trying to detect,” Nieves says. “ACD helped identify unique regions within our drug product and developed custom probes to detect it within tissues.”

Using BaseScope, the team showed that intravitreally delivered ADVM-062 can transduce and express human L-opsin in foveal cone cells. They could also quantify the number of cells expressing the transgene, linking transduction efficiency to expression³.

Achieving orphan drug status would have been difficult without these data, Nieves says. “Using a surrogate vector with a reporter tag to study biodistribution can be useful, but it’s not the product that will go into patients. It’s more powerful to have direct evaluations of the drug product you will use. I think this will be required for all gene therapy packages going forward.”